CN103930165B - To treat CaMKII, IP3R of the metabolic disorder of obesity, calcineurin, P38 and MK2/3 inhibitor - Google Patents

To treat CaMKII, IP3R of the metabolic disorder of obesity, calcineurin, P38 and MK2/3 inhibitor Download PDF

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CN103930165B
CN103930165B CN201280053199.3A CN201280053199A CN103930165B CN 103930165 B CN103930165 B CN 103930165B CN 201280053199 A CN201280053199 A CN 201280053199A CN 103930165 B CN103930165 B CN 103930165B
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camkii
mouse
subject
liver
compound
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CN103930165A (en
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I·塔巴斯
L·厄兹詹
李刚
M·蒙特米尼
王国
王一国
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Columbia University in the City of New York
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Abstract

The method that the method for coronary artery disease and the hepatic glucose of reduction subject of the subject of method, treatment or prevention with metabolic disorder the present invention relates to the metabolic disorder for treating or preventing subject generates.The described method includes but be not limited to the subject apply CaMKII, IP3R, calcineurin, p38, MK2/3 or any combination thereof inhibitor.The method that the present invention also provides the activity of identification inhibition CaMKII, IP3R, calcineurin, p38 or MK2/3 or reduces the compound of the activity and/or activation of CaMKII, IP3R, calcineurin, p38 or MK2/3.

Description

To treat CaMKII, IP3R of the metabolic disorder of obesity, calcium tune nerve phosphoric acid Enzyme, P38 and MK2/3 inhibitor
This application claims the U.S. Provisional Application No. that September in 2011 is submitted on the 2nd to carry on March 30th, 61/530,851,2012 The U.S. Provisional Application No. 61/621,407,2012 that the U.S. Provisional Application No. of friendship is submitted on April 6th, 61/618,551,2012 On July 26, the U.S. Provisional Application No. 61/676,091 the submitted and U.S. Provisional Application No. 61/ submitted on July 26th, 2012 676,152 priority, the provisional application are all incorporated herein in its entirety by reference.
This patent is disclosed containing material protected by copyright.Copyright owner does not oppose by anyone with patent document or special The open United States Patent (USP) that appears in of profit replicates the patent document or patent disclosure with the form in Trademark Office patent file or record, But in addition retain any and all copyright rights whatsoever.
All patents referred to herein, patent application and announcement are incorporated herein in its entirety by reference accordingly.These The disclosure of announcement is incorporated hereby the application to be described more fully wherein such as those of skill in the art accordingly The state of the art on the known date by invention as described herein.
Government-funded
The present invention is according to by National Institutes of Health (National Institute of Health) and state-run heart, lung And the number of the authorizing P01HL087123 that Blood Research Institute (National Heart, Lung and Blood Institute) is issued It is carried out under government-funded.Therefore, U.S. government has certain rights to this invention.
Background of invention
The insulin resistance and hepatic glucose and fat metabolic disturbance that obesity induces increase heart disease, cancer and other wide The risk of cloth and crushing disease.Current therapeutic option is extremely limited, and so as to have an impact, to suffer from current obesity epiphytotics The urgent of hundreds of millions overweight personnel does not meet clinical needs.To treat and diagnose obesity induction insulin resistance and liver There are needs in the method for glucose and fat metabolic disturbance.These needs of present invention processing.
Summary of the invention
The disclosure provides the method for treating and/or preventing metabolic disorder.The disclosure also provides to identify to control The method of the combination of the compound or compound of the metabolic disorder for the treatment of and/or prevention subject.
The disclosure provides the method for treating and/or preventing metabolic disorder.On the one hand, the disclosure provide it is a kind for the treatment of or Prevent the method for the metabolic disorder of subject, wherein the illness is induced by obesity, the described method includes reduce CaMKII's Activity, so as to treat or prevent the illness.In one embodiment, illness is selected from by type 1 diabetes, diabetes B, pancreas The group of island element resistance and metabolic syndrome composition.In another embodiment, treating or preventing influences the decomposition of glycogen of subject Or gluconeogenesis.In one embodiment, the hepatic glucose generation for reducing subject, hyperglycaemia, fatty liver, pancreas are treated or prevented Island element resistance, the relevant inflammation of insulin resistance, the relevant dyslipidemia of insulin resistance or any combination thereof.In another implementation In scheme, the phosphorylation for reducing CaMKII and/or activation are treated or prevented.In one embodiment, the activity of CaMKII is The activity of glucagon (glucagon) induction.In one embodiment, treating or preventing, which reduces G6pc and/or Pck1, exists Expression in the cell of subject.In another embodiment, the FoxO1 in the core for reducing the cell of subject is treated or prevented Protein content.In another embodiment, treat or prevent reduce amino acid S284, S295 of FoxO1 protein, S326, S467, S475, S246, S253, S413 or S415 or any combination thereof phosphorylation.
On the other hand, the disclosure provides a kind of coronary artery disease for treating or preventing the subject with metabolic disorder Method, the described method includes the activity for the CaMKII for reducing the subject, so as to treat or prevent the coronary artery disease. In one embodiment, illness is selected from what is be made of type 1 diabetes, diabetes B, insulin resistance and metabolic syndrome Group.In another embodiment, treating or preventing influences the decomposition of glycogen or gluconeogenesis of subject.In one embodiment, The hepatic glucose generation for the treatment of or prevention reduction subject, hyperglycaemia, fatty liver, insulin resistance, insulin resistance are relevant The relevant dyslipidemia of inflammation, insulin resistance or any combination thereof.In another embodiment, treating or preventing reduces The phosphorylation of CaMKII and/or activation.In one embodiment, the activity of CaMKII is the activity of glucagon induction. In one embodiment, the expression for reducing G6pc and/or Pck1 in the cell of subject is treated or prevented.In another implementation In scheme, the FoxO1 protein contents in the core for reducing the cell of subject are treated or prevented.In another embodiment, control Treat or prevent to reduce amino acid S284, S295, S326, S467, S475, S246, S253, S413 or S415 of FoxO1 protein Or any combination thereof phosphorylation.In one embodiment, treat or prevent in the macrophage for including reducing subject CaMKII activity.
In one embodiment, coronary artery disease is formed to atheroma and/or atherosclerosis is related. In another embodiment, method further comprises treating or preventing heart failure, hypertension and/or nephrosis.In another embodiment party In case, illness is related to advanced lesions macrophage apoptosis.In another embodiment, illness is related to patch necrosis. In another embodiment, treating or preventing the coronary artery disease of the subject with metabolic disorder causes to mitigate hyperinsulinemia Disease and/or dyslipidemia.In another embodiment, the coronary artery disease of the subject with metabolic disorder is treated or prevented Cause to mitigate atheroma formation and/or atherosclerosis.
In one embodiment, the gene for including that coding CaMKII protein is reduced to subject's application is treated or prevented Expression antisense RNA the step of.In another embodiment, treating or preventing includes specifically binding to subject's application The step of peptide or polypeptide of CaMKII protein.In another embodiment, treating or preventing includes applying small point to subject The step of son.In one embodiment, small molecule is the CaMKII inhibitor selected from the group being made up of：KN-93, grass is smoked Rhzomorph C (lavendustin C), CK59, Ant-CaMKIINtide, KN62, DY9760e, Nocardiopsis (Nocardiopsis sp.) K-252a, H89 dihydrochloride, PP1 analog II1NM-PP1, eEF-2 kinase inhibitors NH125 And STO-609.
On the other hand, the disclosure provides a kind of method that hepatic glucose for reducing subject generates, and the described method includes drops The activity of low CaMKII, so as to cause the reduction of the hepatic glucose of the subject.In one embodiment, liver is reduced Glucose generates the phosphorylation for reducing CaMKII and/or activation.In another embodiment, the activity of CaMKII is pancreas hyperglycaemia The activity of element induction.In another embodiment, reduce hepatic glucose and generate the cell for reducing G6pc and/or Pck1 in subject In expression.In one embodiment, the hyperglycaemia, fatty liver, insulin for reducing hepatic glucose generation reduction subject resist Property, the relevant inflammation of insulin resistance, the relevant dyslipidemia of insulin resistance.In another embodiment In, reduce the FoxO1 protein contents in the core for the cell that hepatic glucose generates reduction subject.In another embodiment, Reduce hepatic glucose generate reduce amino acid S284, S295 of FoxO1 protein, S326, S467, S475, S246, S253, S413 or S415 or any combination thereof phosphorylation.
In one embodiment, reducing hepatic glucose and generating includes reducing coding CaMKII protein to subject's application Gene expression antisense RNA the step of.In another embodiment, reducing hepatic glucose and generating includes applying to subject The step of specifically binding the peptide or polypeptide of CaMKII protein.In another embodiment, reducing hepatic glucose generation includes The step of small molecule being applied to subject.In one embodiment, small molecule is the CaMKII selected from the group being made up of Inhibitor：KN-93, lavendustin C, CK59, Ant-CaMKIINtide, KN62, DY9760e, Nocardiopsis K-252a, H89 dihydrochlorides, PP1 analog II1NM-PP1, eEF-2 kinase inhibitors NH125 and STO-609.
On the other hand, the disclosure provides a kind of method of the compound for the activity for inhibiting CaMKII for identification, the side Method includes cell a) is made to contact with CaMKII fusion proteins, wherein the CaMKII fusion proteins are included in the receptor of an end Fluorogen protein and the donor fluorophore protein in another end；And b) in the feelings the absence and presence of test compound FRET efficiency is measured under condition, wherein compared to the FRET efficiency in the case of there is no the test compound, in the survey Try the larger instruction of the FRET efficiency activity tested compound and inhibit CaMKII in the presence of compound.
In one embodiment, acceptor fluorescence group protein is selected from the group being made up of：mOrange、 MStrawberry, Venus, yellow fluorescence protein, cyan fluorescent protein, red fluorescent protein and green fluorescent protein.
In another embodiment, cell be HEK293T cells, liver cell, U2OS cells, HeLa cells, mouse embryo into Fibrocyte (MEF) or macrophage.In another embodiment, cell from Insr-/- mouse, Camk2g-/- mouse, Foxo1-/- mouse, db/db mouse, ob/ob mouse, p38-/- mouse, non-obese diabetes (NOD) mouse, feeding are high in fat The mouse of meals or the mouse of streptozotocin (streptozotocin) processing.In another embodiment, cell is from expression It is mutated the mouse of FoxO1 protein.In one embodiment, be mutated FoxO1 protein be included in S284, S295, S326, At S467, S475, S246, S253, S413 or S415 alanine substitution or S284, S295, S326, S467, S475, At S246, S253, S413 or S415 aspartic acid substitution or any combination thereof.
In one embodiment, cell is made to be subjected to ER stress.In another embodiment, cell glucagon, 8- bromine cAMP, H89 dihydrochloride, smooth Spongistatin C (Xestospongin C), forskolin (forskolin), saturated fatty acid Or any combination thereof processing.
On the one hand, the disclosure provide it is a kind of treat or prevent subject metabolic disorder method, wherein the illness by Obesity induces, the described method includes the activity for reducing IP3R1, IP3R2 or IP3R3, so as to treat or prevent the illness.Separately On the one hand, the disclosure provides a kind of method for the metabolic disorder for treating or preventing subject, wherein the illness is lured by obesity Hair, the described method includes the activity for reducing calcineurin, so as to treat or prevent the illness.
In one embodiment, illness was selected from by type 1 diabetes, diabetes B, insulin resistance, obesity and generation Thank to the group of syndrome composition.
In another embodiment, treating or preventing influences the decomposition of glycogen or gluconeogenesis of subject.
In one embodiment, the hepatic glucose generation for reducing subject, hyperglycaemia, fatty liver, pancreas are treated or prevented Island element resistance, the relevant inflammation of insulin resistance, the relevant dyslipidemia of insulin resistance or any combination thereof.
In one embodiment, treating or preventing includes reducing coding IP3R1 protein, IP3R2 to subject's application The step of antisense RNA of the expression of the gene of protein or IP3R3 protein.In another embodiment, bag is treated or prevented The step of including the peptide or polypeptide to subject application specific binding IP3R1 protein, IP3R2 protein or IP3R3 protein.
In one embodiment, the step for the treatment of or preventing including applying small molecule to subject.In an embodiment party In case, small molecule is IP3R1 protein inhibitors, IP3R2 protein inhibitors or IP3R3 protein inhibitors.
In one embodiment, treating or preventing includes reducing coded Ca tune nerve phosphatase protein to subject's application The step of antisense RNA of the expression of the gene of matter.In another embodiment, treating or preventing is included to subject's application specifically The step of peptide or polypeptide of property combination calcineurin protein.
In one embodiment, the step for the treatment of or preventing including applying small molecule to subject.In another embodiment party In case, small molecule is Calcineurin inhibitors.
On the other hand, the disclosure provides a kind of compound for the activity for inhibiting IP3R1, IP3R2 or IP3R3 for identification Method is contacted the described method includes cell a) is made with test compound；And IP3R1, IP3R2 or IP3R3 activity b) are measured, In activity compared to IP3R1, IP3R2 or IP3R3 in the case of there is no the compound, there are the compounds In the case of the activity of IP3R1, IP3R2 or IP3R3 reduce and indicate that the compound is IP3R1, IP3R2 or IP3R3 respectively Inhibitor.
In one embodiment, by after being stimulated with the derivant of IP3, discharging to the calcium in the cytosol of cell To measure activity.In one embodiment, discharged by the increase of the fluorescence of kytoplasm liquid calcium dyestuff to measure calcium.In a reality It applies in scheme, kytoplasm liquid calcium dyestuff is Fluo-3.
On the other hand, the disclosure provides a kind of side of the compound for the activity for inhibiting calcineurin for identification Method is contacted the described method includes cell a) is made with test compound；And Calcineurin Activities b) are measured, wherein comparing In the calcineurin in the case of there is no the compound activity in the case of there are the compound The activity of calcineurin reduces the inhibitor for indicating that the compound is calcineurin.
In one embodiment, phosphatase activity is detected by using calcineurin peptide substrate to measure calcium tune The activity of neural phosphatase.In one embodiment, cell is HEK293T cells, liver cell, U2OS cells, HeLa cells Or rat embryo fibroblast cell (MEF).In another embodiment, cell is from db/db mouse, ob/ob mouse, non-obese sugar The mouse of sick (NOD) mouse of urine, the mouse of feeding high fat diet or streptozotocin processing.
In one embodiment, cell glucagon, H89 dihydrochlorides, insulin, forskolin, ER stress Derivant, tunicamycin (tunicamycin), saturated fatty acid or any combination thereof processing.
On the other hand, the disclosure provides a kind of method for the metabolic disorder for treating or preventing subject, wherein the illness It is induced by obesity, the described method includes the activity for reducing p38, so as to treat or prevent the illness.On the other hand, the disclosure A kind of method for the metabolic disorder for treating or preventing subject is provided, wherein the illness is induced by obesity, the method bag Including reduces the activity of MK2/3, so as to treat or prevent the illness.
In one embodiment, illness was selected from by type 1 diabetes, diabetes B, insulin resistance, obesity and generation Thank to the group of syndrome composition.
In one embodiment, treating or preventing influences the decomposition of glycogen or gluconeogenesis of subject.In another embodiment party In case, the hepatic glucose generation for reducing subject, hyperglycaemia, fatty liver, insulin resistance, insulin resistance phase are treated or prevented The relevant dyslipidemia of the inflammation of pass, insulin resistance or any combination thereof.
In one embodiment, treating or preventing includes the gene to subject's application reduction coding p38 protein The step of antisense RNA of expression.In another embodiment, treating or preventing is included to subject's application specific binding p38 The step of peptide or polypeptide of protein.In another embodiment, the step for including that small molecule is applied to subject is treated or prevented Suddenly.In one embodiment, small molecule is p38 inhibitor.
In one embodiment, the gene for including that coding MK2/3 protein is reduced to subject's application is treated or prevented Expression antisense RNA the step of.In another embodiment, treating or preventing includes specifically binding to subject's application The step of peptide or polypeptide of MK2/3 protein.In one embodiment, treating or preventing includes applying small molecule to subject The step of.In one embodiment, small molecule is MK2/3 inhibitor.
On the other hand, the disclosure provides a kind of method of the compound for the activity for inhibiting p38 for identification, the method bag Including a) makes cell be contacted with test compound；And p38 kinase activities b) are measured, wherein compared to there is no the compounds In the case of p38 kinase activity, the kinase activity of the p38 in the case of there are the compound reduces instruction describedization Close the inhibitor that object is p38.On the other hand, the disclosure provides a kind of side of the compound for the activity for inhibiting MK2/3 for identification Method is contacted the described method includes cell a) is made with test compound；And b) measure MK2/3 kinase activities, wherein compared to The kinase activity of MK2/3 in the case of there is no the compound, MK2/3's in the case of there are the compound swashs Enzymatic activity reduces the inhibitor for indicating that the compound is MK2/3.
In one embodiment, p38 kinase activities are measured using p38 specific peptides.In one embodiment, use MK2/3 specific peptides measure MK2/3 kinase activities.
In one embodiment, cell is HEK293T cells, liver cell, U2OS cells, HeLa cells, macrophage Or rat embryo fibroblast cell (MEF).In another embodiment, cell is from Insr-/- mouse, db/db mouse, ob/ob The mouse that mouse, non-obese diabetes (NOD) mouse, the mouse of feeding high fat diet or streptozotocin are handled.In another reality It applies in scheme, cell glucagon, 8- bromine cAMP, H89 dihydrochloride, forskolin, saturated fatty acid or any combination thereof Processing.
These and other embodiment of the present invention is together with will be by more when being described below and considering with alterations It is appreciated and understood by well.It should be appreciated, however, that although instruction various embodiments of the present invention and its numerous specific details, it is following Description is only provided by illustrative and not limiting mode.It can be permitted within the scope of the invention without departing from the spirit of the present invention It is substitute, modification, addition and/or reset more, and the present invention includes all such substituting, modification, addition and/or resetting.
Brief description
Figure 1A-J. glucagons and fasting activation liver CaMKII.Figure 1A with 100nM glucagons (Gluc) or Intermedium control (Veh) stimulate measured in the triplicate hole of the Primary mouse HC of instruction time CaMKII enzymatic activitys (compared with Veh*P<0.05 and * * P<0.01；Average value ± S.E.M.).Figure 1B -1J. measure the extraction of detection HC or liver by Western blotting Phosphoric acid-CaMKII, total CaMKII and the beta-actin of object.When Figure 1B make HC and the 00nM glucagons be incubated with instruction Between；In HC when glucagon is added to intermedium control (Veh) or small 5 μM of BAPTA-AM pretreatments 1 by Fig. 1 C.；Figure Glucagon is added to the HC pre-processed with 0.5 μM of smooth Spongistatin (XesC) or is added to-LacZ pairs of gland of using by oneself by 1D. According to or gland-Cre transductions Ip3r1fl/fl mouse HC in (bar chart=Ip3r1mRNA horizontal)；Fig. 1 E. are by glucagon In HC during added to intermedium control (Veh) or small 10 μM of H89 pretreatments 1.Fig. 1 F. make HC and 100 μM of bromo- cAMP of 8- It is incubated with the instruction time.Fig. 1 G-1J. experiment in vivo.In figure 1g, the glucagon abdomen of 200 μ g kg-1 weight of mouse (i.p.) is handled 30 minutes in film, and in Fig. 1 H, mouse uses the smooth sponges of 10pmol g-1 before glucagon processing It is pre-processed 4 days in plain C or intermedium control peritonaeum.In Fig. 1 I-1J, mouse is by when arbitrary feeding or small fasting 12 or fasting 12 it is small when and when then feeding 4 is small again.
Glucose in Fig. 2A-E.CaMKII regulation and control primary hepatocytes generates and liver G6Pc and Pck1 expression.Fig. 2A is in MOI For 20 times adenoviral vector transduction Primary mouse liver cells with expression LacZ, CA-CaMKII or K43A-CaMKII.Make cell Be subjected to staying overnight serum exhaust and be then incubated in the culture medium of serum-free and glucose with forskolin (10 μm) 14 it is small when. Such as the measurement glucose content.Fig. 2 B are as in Fig. 2A, with the exception is that thin using WT liver cells and Camk2g-/- liver Born of the same parents.Fig. 2 C use the adenovirus vector infector for expressing LacZ, CA-CaMKII or K43A-CaMKII for Mouse Liver in MOI for 20 times Cell.Make cell be subjected to staying overnight serum exhaust and be then incubated in serum free medium with forskolin (10 μm) 5 it is small when. It separates RNA and passes through real-time Q-PCR analysis gene expression.(*, P<0.05；*, P<0.01) Fig. 2 D make from WT mouse and The primary hepatocyte of Camk2g-/- mouse be subjected to stay overnight serum exhaust and then in serum free medium with forskolin (10 μm) Be incubated with 5 it is small when.G6pc and Pck1mRNA in RNA is measured by real-time Q-PCR.(* *, P<0.01；* * *, P< 0.0005).Fig. 2 E. are as in Fig. 2 D, with the exception is that glucagon (100nm) is used to stimulate gluconeogenesis.(*, P< 0.05；*, P<0.01)
Liver FoxO1 Subcellular Localizations in Fig. 3 A-C.CaMKII regulation and control primary hepatocytes.Fig. 3 A are used for 2 times in MOI and expressed Primary hepatocyte of the adenoviral transduction of mouse GFP-FoxO1 from WT mouse and Camk2g-/- mouse.Cell is made to be subjected to staying overnight blood It is clear exhaust and be then incubated in serum free medium 5 it is small when.FoxO1 Subcellular Localizations are assessed by indirect immunofluorescence.Than Example ruler (bar), 10 μm.The quantitative data in right figure.(* * * *, P<0.0005).Fig. 3 B are used for 20 times in MOI and are expressed LacZ, CA- The adenoviral vector transduction Primary mouse liver cell of CaMKII or K43A-CaMKII.4 it is small when after, in MOI for 5 time with mouse GFP- FoxO1 adenovirus infected cells.Make cell be subjected to stay overnight serum exhaust and then in serum free medium be incubated 5 it is small when.With FoxO1 Subcellular Localizations are assessed by indirect immunofluorescence afterwards.Engineer's scale, 5 μm.The quantitative data in figure below.(* * * *, P< 0.0005；####P<0.0005).Fig. 3 C Primary mouses liver cell is infected with the adenovirus vector of expression LacZ or CA-CaMKII. The FoxO1 of nuclear extract and phosphocarnic acid albumen (core loads control) are measured by Western blotting.
Fig. 4 A-D.CaMKII γ, which lack, reduces blood glucose, G6pc and Pck1 expression and core FoxO1.8 when Fig. 4 A fasting 12 is small Blood sugar concentration (*, P in week old WT mouse and Camk2g-/- mouse<0.05) Fig. 4 B by 8-10 week old WT mouse and It is excited when Camk2g-/- mouse fasting 18 is small and then with pyruvic acid (2mg kg-1).(*, P<0.05；*, P<0.01；* *, P <0.005) liver G6pc and the Pck1 gene expression dose in WT mouse and Camk2g-/- mouse when Fig. 4 C fasting 12 is small.(* *, P<0.01；* *, P<0.001) Fig. 4 D are as in Fig. 4 C, with the exception is that measuring liver nuclear extract by Western blotting FoxO1 and phosphocarnic acid albumen.The densitometry quantitative display of these data is in figure below.(* * *, P<0.001)
The acute inhibition CaMKII of Fig. 5 A-F. reduce blood glucose, G6pc and Pck1 expression and core FoxO1.It is used by tail vein The 9 week old WT mouse of adenoviral injection containing control LacZ (n=5) or K43A-CaMKII (n=5) of 1.5x109pfu.Fig. 5 A The 5th day when fasting 12 is small after blood glucose level.(* * *, P<0.001) glycogen in mouse when Fig. 5 B fasting 12 is small is different Raw gene expression.(* *, P<0.01；* *, P<0.001).Fig. 5 C. are as in Fig. 5 B, with the exception is that passing through Western blotting Measure the FoxO1 of liver nuclear extract and phosphocarnic acid albumen.Fig. 5 D hepatic glycogen contents.The pancreas of 200 μ g kg-1 weight of Fig. 5 E-5F Glucagons peritonaeum Inner injects the mouse of LacZ or K43A-CaMKII processing and puts to death after 30 minutes.Measure liver G6pc MRNA, and by the glycogen content of PAS chromoscopy liver slices, (n=4 is only small for the percentage of quantification of PAS positive hepatocellulars Mouse).Engineer's scale, 20 μm.
Fig. 6 A-D. constitutive activities CaMKII increases blood glucose, G6pc and Pck1 expression and core FoxO1.Use 1.5x109pfu Containing control LacZ (n=5) or CA-CaMKII (n=5) 9 week old WT mouse of adenoviral injection.Fig. 6 A were prohibiting at the 6th day Eat 12 it is small when after measure blood glucose.(*, P<0.05) pyruvic acid challenge test is carried out in mouse of Fig. 6 B when fasting 17 is small.(*, P<0.05；*, P<0.01；* *, P<0.005) (*, P when the fasting 12 of Fig. 6 C measure is small<Or arbitrary feeding (*, P 0.05)< 0.05；* *, P<0.005) the G6pc and Pck1mRNA levels in mouse.Fig. 6 D are measured by Western blotting with control LacZ (n=5) or CA-CaMKII (n=5) injection feeding mouse in the FoxO1 of liver nuclear extract and phosphocarnic acid albumen.
In Fig. 7 A-D.Camk2g-/- liver cell and internal glucose metabolism damage with composing type core FoxO1-ADA by being transduceed And it is saved.Fig. 7 A MOI be 0.2 time with the adenoviral transduction for expressing FoxO1-ADA come from WT mouse and Camk2g-/- it is small The primary hepatocyte of mouse.Cell is made to be subjected to staying overnight serum exhaustion and then in serum free medium together with forskolin (10 μm) Be incubated 5 it is small when.It separates RNA and passes through real-time Q-PCR analysis gene expression.(* *, P<0.01；#, P<0.05；##, P<0.01) figure 7B 1.5x109The 8 week old WT of adenoviral injection containing control LacZ (n=5) or K43A-CaMKII (n=10) of pfu is small Mouse.After 1 day, the mouse of half gland-K43A-CaMKII injections receives 0.1x109The adenovirus containing FoxO1-ADA of pfu, and Remaining mouse receives LacZ controls.The 5th day when fasting 12 is small after measure blood glucose level.(*, P<0.05；#, P<0.05) Fig. 7 C measure mouse of FoxO1 and phosphocarnic acid albumen Fig. 7 the D measure of liver nuclear extract from fasting 12 when small by Western blotting Liver G6pc, Pck1 and Igfbp1mRNA it is horizontal.(*, P<0.05；*, P<0.01；#, P<0.05)
P-CaMKII γ in Fig. 8 A-B. obesity livers.From Fig. 8 A10 week old WT mouse or ob/ob mouse and feeding standard Meals or high calorie (DIO) 20 weeks mouse of meals；Or the Western blotting of the liver extract of Fig. 8 B laths or morbid obesity people. The densitometry of mouse data in display Fig. 8 A quantifies；P<0.001.
In obesity mice, CaMKII γ lack mitigation hyperinsulinemia and improve insulin and glucose Fig. 9 A-I. Metabolism.Plasma insulin in Fig. 9 A-9B, feeding and fasting WT (n=10) and Camk2g-/- (n=7) DIO- obese male mouse Element.Insulin resistance test (ITT) in Fig. 9 C, WT mouse and Camk2g-/- mouse (n=5).Fig. 9 D, weight.Fig. 9 E- G, before or after with gland-LacZ controls or gland-K43ACaMKII (dominant negative) processing, the plasma insulin in DIO mouse The liver mRNA (n=6) of element, glucose tolerance test (GTT) and FoxO1 targets.In fig. 9e, *, P<0.01 comparison adenovirus It is preceding and<0.005 the 7th day LacZ of comparison.In Fig. 9 G, *, P<0.01.Fig. 9 H-I, ob/ob obesity mice CaMKII inhibitor KN93 or its inactivation homologue KN92 processing.Then measuring the expression of blood glucose and G6pc and Pck1 in liver, (repetition values become Change 10%).
Figure 10 A-D.CaMKII γ lack steatosis, dyslipidemia and the inflammation improved in obesity mice.Figure 10 A-B, 10D after DIO meals 20 weeks, measures WT (n=10) and liver TG contents in Camk2g-/- (n=7) mouse, blood plasma lipide With liver TnfamRNA (G, *, P<0.005；H, *, P<0.05**, P<0.01；I, *, P<0.05).Figure 10 C, with-LacZ pairs of gland According to or gland-K43ACaMKII (dominant negative) processing before or after, p-S6K and total S6K in the liver from DIO mouse and The Western blotting of sorting protein -1.
Figure 11 .CaMKII γ lack the containment mediated by added machinery to atherosclerosis.
Figure 12 .CaMKII γ are induced in UPREffect in Apoptosis
Figure 13 .CaMKII γ haploinsufficiencies are protectedExempt from the Apoptosis of ER stress-induceds.It is filled in control or FC Carry under the conditions of (cholesterol) processing from WT, Camk2g+/- or Camk2g-/- mouseAnd then measure Apoptosis. *, # ,@=P relative to each other<0.01.
ER stress-induceds of Figure 14 .CaMKII γ in insulin resistance environmentKey effect in Apoptosis General introduction.
Figure 15 A-B.Insulin resistance and ERK1/2 inhibit to increase p-CaMKII.Figure 15 A WT, Insr-/- and ob/ ob ± FC is loaded or oxLDL and Figure 15 B handle WT with 5 μM of U0126ERK inhibitor± 4 it is small when pThr287- The Western blotting of CaMKII.
Figure 16 .CaMKII γ lack containment UPR.Make from WT mouse or Camk2g-/- mouseWith thapsigargin (Thaps) be incubated together or under FC loading conditionses (cholesterol) and then carry out Western blotting with measure UPR protein, CaMKII γ and beta-actin.
P-CaMKI in Figure 17 late period human coronary artery spots macrophage in the block.The area of inspection is by the Movat five colors Block delineations in the patch sample of staining reagent；NC, necrotic cores；Engineer's scale, 2mm.It is if positive, then antibody is anti- Micro- rufous product should be generated, and if negative, then it is light blue.For enlarged drawing, engineer's scale, 100 μm.
Figure 18 liver cell CaMKII γ lack by via be related to the mechanism that FoxO1 is arranged outside core reduce gluconeogenesis (GNG) and Hepatic glucose generates (HGP) to mitigate hyperinsulinemia and subsequent dyslipidemia.
Figure 19 A-H. fasting activates liver CaMKII, and CaMKII lacks or inhibit containment GNG and core FoxO1.Figure 19 A, WT Mouse is by (top) when arbitrary feeding or small fasting 12；Or fasting 12 it is small when and when then feeding 4 is small again (lower part).Liver is extracted Object carries out Western blotting to measure p-CaMKII, total CaMKII and beta-actin.Figure 19 B, WT mouse when fasting 12 is small and Blood glucose (*, P in Camk2g-/- mouse or the WT mouse handled with gland-LacZ or gland-K43A-CaMKII<0.001).Figure 19C, the blood glucose in WT mouse and Camk2g-/- mouse when the fasting 18 excited with 2mg/kg pyruvic acid is small.Figure 19 D, with gland- Liver G6pc and Pck1mRNA (*, P in the mouse of LacZ or gland-K43A-CaMKII processing<0.01).Figure 19 E, to coming from B The nuclear extract of liver of experiment (left side) carry out Western blotting to measure FoxO1 and phosphocarnic acid albumen (Np；Core loads control) And (*, P are quantified by densitometry art<0.001).Figure 19 F, the primary HC from WT mouse or Camk2g-/- mouse are (left Side)-or with gland-LacZ, constitutive activity CaMKII (T287D) or K43A-CaMKII transduce WT HC (right side)-with gland- (common) transductions of GFP-FoxO1.It is quantified by the graphical analysis to immunofluorescence confocal microscopy data in serum starved cells FoxO1 Subcellular Localizations (*, #, P<0.005).Figure 19 G, the HC from WT mouse or Camk2g-/- mouse with gland-LacZ or FoxO1-ADA transduces, and is then subjected to serum starvation, is handled with forskolin, and measures G6pc mRNA (*, P<0.01).Figure 19 H, The Ser residues being phosphorylated in the HC lacked in serum starvation WT HC rather than in CaMKII；DBD=DNA binding structural domains.
In the liver of Ldlr-/- mouse of WD feedings, CaMKII γ are phosphorylated Figure 20 A-B..It is measured by Western blotting With blood glucose (Figure 20 A of standard diet or the Western diet Ldlr- of 11 weeks/- mouse；P<0.001) it is and liver p-CaMKII, total CaMKII and beta-actin (Figure 20 B).
The molecule that the hepatic glucose of glucagon induction when Figure 21 A-21AV are shown in fasting and obesity generates Association.During Figure 21 A are shown in hepatic glucose generation, the direct signal conduction of decomposition of glycogen and gluconeogenesis and transcriptional control.Figure 21B shows that hepatic glucose generates the transcription control of (HGP).Figure 21 C show hepatic glucose generation, fasting/glucagon, pancreas islet Plain resistance/ associating between DM2 and calcium and obesity.Research calcium is increased and associating between HGP.Figure 21 D-F show calcium, liver Glucose generates the associated brief history viewpoint between Apoptosis.Figure 21 D are shown in advanced atherosclerosis The figure of macrophages die.Figure 21 E are display calcium sensitivity signal transduction molecule calcium/Calmodulin Dependent Protein Kinases II (CaMKII) figure.Figure 21 F are the figures for the mechanism for showing the macrophages die in atherosclerosis.Figure 21 G are shown in Under without being bound by theory, the figure of the controllable HGP of CaMKII- γ.Figure 21 H-L show the primary mouse liver of glucagon activation culture CaMKII in cell.Figure 21 H show that glucagon activates the CaMKII in primary hepatocytes.Figure 21 I show the high blood of pancreas Associating between sugared element and the activation of CaMKII.Figure 21 J show associating between glucagon and calcium increase.Figure 21 K are shown Glucagon activates liver CaMKII by IP3R in vivo.Figure 21 L show the activation of fasting in vivo and again feeding containment liver CaMKII.Figure 21 M show that the glucose reached by primary hepatocyte that CaMKII is induced in PKA plays an important role in generating.Figure 21N shows in Gcpc and the Pck1 induction of PKA and glucagon mediation of the CamKII in primary hepatocyte and plays an important role. Figure 21 O are to show the mechanism induced by gene, associated between fasting and glucagon, CaMKII and glucose generation Figure.Figure 21 P are the figures for the transcription control for showing HGP.Figure 21 Q are shown in serum starvation primary hepatocyte, and FoxO1 is in core In.Figure 21 R show in FoxO1 nuclear locations of the CamKII in serum starvation primary hepatocyte and play an important role.Figure 21 S are shown CamKII plays an important role in the FoxO1 nuclear locations in serum starvation primary hepatocyte.Figure 21 T show the albumen of nuclear extract Matter trace measures, and display CaMKII plays an important role in the FoxO1 nuclear locations in serum starvation primary hepatocyte.Figure 21 U It is the figure for the transcription control for showing the HGP including CRTC2-PGC1 α approach.Figure 21 V show CaMKII not in primary hepatocyte Pgc1 induction CRTC2 nuclear locations in work.Figure 21 W are display fasting and glucagon generation, CaMKII, FoxO1 Associated figure between being generated with glucose.Fasting CaMKII γ are presented in Figure 21 X-/-Blood glucose and pyruvic acid excitation in mouse are surveyed The internal evidence of examination.Fasting CaMKII γ are presented in Figure 21 Y-/-The internal card of liver Gcpc/Pck1mRNA and core FoxO1 in mouse According to.The internal evidence of the blood glucose and glycogen storage cavern in the WT mouse of fasting gland-K43ACaMKII transductions is presented in Figure 21 Z.Figure 21 AA The internal evidence of the liver Gcpc/Pck1mRNA and core FoxO1 in the WT mouse of fasting gland-K43A CaMKII transductions is presented.Figure Liver Gcpc mRNA and glycogen storage cavern in the WT mouse of the gland-K43ACaMKII transductions of glucagon processing is presented in 21AB Internal evidence.The internal of blood glucose in the WT mouse of fasting gland-CA CaMKII transduction and pyruvic acid challenge test is presented in Figure 21 AC Evidence.The internal of the liver Gcpc/Pck1mRNA in the WT mouse of fasting and the gland-CA CaMKII transductions of feeding is presented in Figure 21 AD Evidence.The internal evidence of the core FoxO1 in the WT mouse of fasting and the gland-CA CaMKII transductions of feeding is presented in Figure 21 AE.Figure 21AF-AH shows the reason for containment effect that CaMKII lacks to HGP is by arranging FoxO1 mediations outside core evidence.Figure 21 AF are Under without being bound by theory, the effect that CaMKII lacks can transduce the figure prevented by using composing type core FoxO-ADA for instruction. Figure 21 AG show the G6pc/Pck1 in the primary hepatocyte of forskolin processing.Figure 21 AH show that fasting gland-K43A CaMKII turn The opposite mRNA level in-site of blood glucose level and blood glucose and FoxO1 genes in the mouse led.Figure 21 AI are display CaMKII mediations Pass through FoxO1 regulate and control glucose generate and obesity and the correlation of insulin resistance figure.Figure 21 AJ are shown in fat small In the liver of mouse, CaMKII γ are activated.Figure 21 AK are shown in the liver of obese people, and CaMKII γ are activated.Figure 21 AL are shown in In obesity mice, CaMKII γ, which lack, reduces plasma insulin and improvement insulin sensitivity.Figure 21 AM are shown in obesity mice In, CaMKII γ lack the response for improving liver to acute insulin.Figure 21 AN-AO show acute adenovirus mediated CaMKII γ Inhibit to improve the metabolizing parameters in ob/ob mouse.Figure 21 AP show that acute adenovirus mediated CaMKII γ inhibit to reduce G6pc With expression of the Pck1 in the liver of ob/ob mouse.Figure 21 AQ show that acute adenovirus mediated CaMKII γ inhibit to reduce ob/ob Core FoxO1 in the liver of mouse.Figure 21 AR show that CaMKII γ lack the fatty liver mitigated in obesity mice.Figure 21 AS are shown CaMKII inhibits to reduce the liver fat generation gene expression in ob/ob mouse.Figure 21 AT show that CaMKII γ lack part and reduce Plasma cholesterol and triglycerides in obesity mice.Figure 21 AU show that CaMKII γ lack the liver TNF reduced in obesity mice α.The working model that the glucose of Figure 21 AV display regulation and control glucagon inductions generates.Future studies include but not limited to HGP How mechanism, the CaMKII shortages of direct measure (clamp research), CaMKII regulation and control cores FoxO1 with glucose utilization can influence Liver ER stress and vice versa, CaMKII lacks there is what to influence, in Kupffer cell insulin sensitivity Whether CaMKII works in obesity, the treatment potentiality of influences and CaMKII of the CaMKII to atherosclerosis.
The glucose that Figure 22 A-E.CaMKII regulate and control in primary HC generates and liver G6Pc and Pck1 expression.Figure 22 A pass through RT- PCR detections are from 3 WT mouse and 3 Camk2g-/-The finger of the RNA of the RNA of the HC of mouse and mouse brain from WT mouse Show Camk2 hypotypes mRNA.Figure 22 B make from WT mouse and Camk2g-/-The HC of mouse is subjected to staying overnight serum exhaustion and then in nothing Be incubated in serum and dextrose culture-medium with forskolin (10 μm) 14 it is small when, and then measure the glucose in culture medium (compared with WT**P in each group<0.01；Average value ± S.E.M.).HCs of Figure 22 C from WT mouse expression LacZ, CA- The adenovirus vector of CaMKII or KD-CaMKII MOI for 20 time transductions and then as measure in Figure 22 B glucose generation ( Compared with LacZ*P in each group<0.05 and * * P<0.01；Average value ± S.E.M.).Figure 22 D-22E make in Figure 22 B and Figure 22 C The similar HC of those HC be subjected to staying overnight serum and exhaust and then as indicated, in serum free medium with 10 μM of forskolins or 100nM glucagons be incubated with 5 it is small when.The G6pc and Pck1mRNA of RNA are measured (in each group relatively by RT-qPCR In LacZ or WT*P<0.05 and * * P<0.01；Average value ± S.E.M.).
CaMKII γ lack in Figure 23 A-G. bodies or acute inhibition reduces blood glucose and liver G6pc and Pck1.Figure 23 A fasting 12 The 8 week old WT mouse of hour and Camk2g-/-Blood glucose (the * P of mouse<0.05).Figure 23 B are as in Figure 23 A, but by mouse Use when fasting 18 is small and then 2mg kg-1Pyruvic acid excites (Figure 23 B).(*P<0.05；**P<0.01；***P<0.005；It is average Value ± S.E.M.).WT mouse and Camk2g when Figure 23 C fasting 12 is small-/-Liver G6pc and Pck1mRNA (* * P in mouse< 0.01；***P<0.001；Average value ± S.E.M.).Figure 23 D glucagon (200 μ g kg-1) the interior injection WT mouse of peritonaeum And Camk2g-/-It mouse and puts to death after 30 minutes.Measure liver G6pc mRNA (* P<0.05；Average value ± S.E.M.).Figure 23E-G applies 1.5x10 to 9 week old WT mouse9Gland-the LacZ or KD-CaMKII of pfu, and after 5 days measure fasting 12 it is small when Following parameter in mouse：Figure 23 E, blood glucose (* * * P<0.001；Average value ± S.E.M.)；Figure 23 F, liver G6pc and Pck1mRNA (**P<0.01；***P<0.001；Average value ± S.E.M.)；With Figure 23 G, hepatic glycogen content and PAS positive cells (* * P< 0.01；Average value ± S.E.M.).Figure G also shows fasting WT mouse and Camk2g-/-Hepatic glycogen content (* P in mouse<0.05； Average value ± S.E.M.).For all figures, in addition to the wherein figure D of n=4/ groups, n=5/ groups.
Figure 24 A-D.CaMKII regulate and control liver FoxO1 Subcellular Localizations.Figure 24 A are used for 2 times in MOI and are expressed mouse GFP-FoxO1 Adenoviral transduction come from WT mouse and Camk2g-/-The HC of mouse.Cell is made to be subjected to staying overnight serum exhaustion and then in serum-free When incubation 5 is small in culture medium.FoxO1 Subcellular Localizations are assessed by indirect immunofluorescence.Engineer's scale, 10 μm.It is fixed in right figure Measure data.(#P<0.0005；Average value ± S.E.M.).Figure 24 B are used for 20 times in MOI and are expressed LacZ, CA-CaMKII or KD- It the adenoviral vector transduction HC of CaMKII and is then transduceed after four hours with gland-GFP-FoxO1, then as carried out in Figure 24 A Fluorescent observation is with quantitative (compared with LacZ#P<0.005；Average value ± S.E.M.).Engineer's scale, 5 μm.Figure 24 C such as Figure 24 B In, with gland-LacZ or CA-CaMKII and then with gland-GFP-FoxO1 transductions HC.It is incubated overnight in serum exhausts culture medium And then in serum free medium be incubated 5 it is small when after, by cell with 100nM insulin handle instruction the time.In Figure 24 B Quantitative FoxO1 Subcellular Localizations are (compared with LacZ*P in each group<0.005；Average value ± S.E.M.).Figure 24 D pass through immune Core FoxO1 and phosphocarnic acid albumen in liver of the trace detection from following mouse：Fasting WT mouse, Camk2g-/-Mouse or use The WT mouse of gland-LacZ or KD-CaMKII processing；The feeding WT mouse handled with gland-LacZ or CA-CaMKII；Or with 200 μ g The feeding WT mouse of processing 30 minutes in the glucagon peritonaeum of kg-1 weight.It is tested for glucagon, average FoxO: Np densitometry ratios (* P=0.029 in figure；Flaw in swimming lane 6 and 8 is analyzed from densitometry to be excluded).
The damage that Figure 25 A-E.CaMKII inhibit to glucose metabolism is drawn by being transduceed with composing type core FoxO1-ADA It rescues.Figure 25 A HCs of the gland-LacZ or CA-CaMKII transductions from wild-type mice or L-FoxO1 knock-out mices.Pass through cell Exhausted by overnight serum and then be not present or there are forskolin (10 μm) in the case of be incubated in serum free medium it is 5 small When.Measure G6pc mRNA (the * P of RNA<0.001；Average value ± S.E.M.).Figure 25 B are in the case where MOI is 0.2 to from WT mouse And Camk2g-/-The HC of mouse applies gland-LacZ or FoxO1-ADA.Cell is made to be subjected to staying overnight serum exhaustion and then in serum-free Be incubated in culture medium with 10 μM of forskolins 5 it is small when.The G6pc and Pck1mRNA of RNA are measured (compared with WT group * * P<0.01； Compared with Camk2g-/-/ LacZ groups#P<0.05 and##P<0.01；Average value ± S.E.M.).Illustration, by immunoblot detection come From the FoxO1 of the core of one group of parallel cell and phosphocarnic acid albumen；Averag density metering is than seeming less than each pair of swimming lane.Figure 25 C-E Apply gland-LacZ or KD-CaMKII to 8 week old WT mouse, and then after 1 day, half gland-KD-CaMKII mouse receive gland- FoxO1-ADA, and in addition half receives gland-LacZ controls.The 5th day when fasting 12 is small after to measure blood glucose level (opposite In LacZ/LacZ*P<0.05；Compared with KD/LacZ#P<0.05；N=5/ groups；Average value ± S.E.M.), and measure the core of liver FoxO1 protein is (compared with KD/LacZ**P<0.01；Average value ± S.E.M.)；G6pc, Pck1 and Igfbp1mRNA are (opposite In LacZ/LacZ*P<0.05 and * * P<0.01；Compared with KD/LacZ#P<0.05；N=3/ groups；Average value ± S.E.M.).Scheme E Illustration show hemagglutinin (HA) mark KD-CaMKII protein level (anti-HA Western blottings).
Effect in the FoxO1 nuclear locations that the non-AKT phosphoric acid site of Figure 26 A-C.FoxO1 is mediated in CaMKII.Figure 26 A exist MOI comes from WT mouse and Camk2g for 2 times with gland-FLAG-FoxO1 transductions-/-The HC of mouse.Cell is made to be subjected to staying overnight serum consumption Exhaust and then in serum free medium be incubated 5 it is small when.Using anti-FLAG Immunological purifications FoxO1, then reduced, be alkylated And proteolytic digestion.As described in experimental arrangement, pass through TiO2Chromatograph enrichment Phosphorylated Peptide and then by LC-MS/MS into Row analysis.Table shows the spectrum count value of the Phosphorylated Peptide in KO and WT samples, Debunker score and A score；Peptide sequence In Δ instruction phosphorylation site.The cutoff value of spectrum count value, Debunker score and A score is separately positioned on 5,0.5 Under 12.Score (italics) is checked manually less than the spectrum of the peptide of these values to eliminate uncertain phosphorylation site (figure 31A-B is directed to WT peptides 4 and 5；And the http being incorporated herein in its entirety by reference://fields.scripps.edu/ Published/foxo1_Tabas_2012/ is for KO peptides 7,10 and 11).The combination spectrum only calculated in KO and WT counts>10 Peptide KO/WT spectrum count ratio.The expression plasmid of Figure 26 B coding mouse Flag-FoxO1 or Flag-7A-FoxO1 mutant Transfect the HC from L-FoxO1 mouse.After 48 hours, cell is made to be subjected to staying overnight serum exhaustion and then in free serum culture Be incubated in base with glucagon (100nm) 4 it is small when.The Flag of nuclear extract and phosphocarnic acid egg are measured by Western blotting (core loads control) in vain, and pass through the Foxo1mRNA of RNA of the RT-qPCR detections from one group of parallel cell.MRNA and Diagnosis of Sghistosomiasis The densitometry quantitative display of mark data (* P in figure<0.005；Average value ± S.E.M.) Figure 26 C are similar with Figure 26 B, exception Part is 1 day after with WT or mutation FoxO1 plasmid transfections, with gland-LacZ or CA-CaMKII transduction HC (* P=0.003； Average value ± S.E.M.).
Figure 27 A-D.CaMKII in obesity hepatic glucose metabolism in action diagram 27A by immunoblot detection come From 10 week old WT mouse or ob/ob mouse or feeding standard diet or high calorie diet the WT mouse of 20 weeks, (meals induce Obesity；DIO the p-CaMKII γ of liver extract) and total CaMKII γ and beta-actin.Densitometry quantitative display (the * * * P in bar chart<0.001；Average value ± S.E.M.).By coming from DIOCamk2g-/-It is not deposited in the liver extract of mouse Display antibody specificity is brought in p-CaMKII γ and total CaMKII γ items.Figure 27 B-D are at gland-LacZ or KD-CaMKII Before or after reason, fasting blood-glucose in ob/ob mouse, the blood glucose after pyruvic acid excitation and liver G6pc, Pck1 and Igfbp1mRNA (n=5/ groups；Compared with LacZ*P<0.05, * * P<0.01, * * * P<0.005 and * * * * P<0.001；Average value ±S.E.M.)。
Figure 28 A-G. (related to Fig. 1 and 2 2).In the glucagon peritonaeum of Figure 28 A mouse dose indicatings injection and It is put to death after 15 minutes.Pass through the phosphoric acid CaMKII and total CaMKII and beta-actin of immunoblot detection liver.Figure 28 B are small Mouse (n=5/ groups) when arbitrary feeding or small fasting 12 and is then measured glucagon (the * P=0.007 of blood plasma；Average value ±S.E.M.).Figure 28 C mices continue 4 days daily with 10pmol g-1In smooth Spongistatin C (XesC) or intermedium control peritonaeum Injection and then by arbitrary feeding or small fasting 12 when.Liver phosphoric acid CaMKII, total CaMKII and β-flesh are measured by Western blotting Filamentous actin is horizontal.Figure 28 D are compared with CA-CaMKII, with the Western blotting of the CaMKII in the HC of gland-LacZ transductions.Figure 28 E HC is transduceed with gland-LacZ or KD-CaMKII.Cell is made to be subjected to staying overnight serum exhaustion and then measures CaMKII activity (* P< 0.005；Average value ± S.E.M.).Figure 28 F are compared with Camk2g-/-Mouse, G6pc in the untreated HC from WT mouse and Pck1mRNA levels (* P0.05).MRNA level in-site in the WT HC handled compared to forskolin in Figure 22 D, in untreated WT HC G6pc and Pck1 level reduce about 165 times and about 2500 times respectively.In Figure 28 G such as Figure 28 D, from Camk2g-/-Mouse HC is transduceed with gland-LacZ or KD-CaMKII, when incubation 14 is small in culture medium ± forskolin, and then measures G6pc mRNA (compared with other 2 groups of * P<0.05；Average value ± S.E.M.).Black circles represent the gland-LacZ WT HC's of forskolin processing G6pcmRNA values (1.99 ± 0.3；Compared with Camk2g-/-Value * P<0.001；Average value ± S.E.M.).
Figure 29 A-E. are related to Figure 23.Figure 29 A measure WT mouse and Camk2g-/-Mouse and with gland-LacZ or KD- Empty stomach glucagon and insulin level in the blood plasma of the WT mouse (n=5/ groups) of CaMKII processing；N.s., not significantly. Figure 29 B make to use by oneself CaMKII of liver of mouse of gland-LacZ or KD-CaMKII processing are immunized precipitation (IP) and then measure CaMKII activity.Data in illustration are by showing Camk2g-/-Kinase activity in the liver of mouse can be neglected, and when nonimmune When IgG is in the CaMKII IP sections of scheme, kinase activity can be neglected to verify that CaMKII measures (* P<0.006；Average value ±S.E.M.).Figure 29 C-DWT mouse gland-LacZ or CA-CaMKII processing (n=5/ groups).After 5 days, measure mouse and exist Blood glucose before or after pyruvic acid excitation, and then after arbitrary one period of feeding, put to death mouse, and measure liver G6pc and Pck1mRNA (* P<0.05；**P<0.01；* *, P<0.005；Average value ± S.E.M.).Figure 29 E mouse with gland- LacZ or CA-CaMKII processing (n=5/ groups).After 5 days, the glycogen content (* of liver is measured mouse overnight fasting and then P<0.05；Average value ± S.E.M.).
Figure 30 A-D. are related to Figure 24-25.Figure 30 A are in MOI for 1 time with gland-LacZ or CA-CaMKII transduction HC and 4 It is transduceed after hour with gland-GFP-FoxO1.It is incubated overnight in serum exhausts culture medium and is then incubated in serum free medium Educate 5 it is small when after, cell with 100nM insulin handle 15 minutes.Quantitative FoxO1 Subcellular Localizations.(*P<0.005；Average value ±S.E.M.).Illustration moves egg by the CaMKII and β-flesh of lysate of the immunoblot detection from one group of parallel cell In vain.Figure 30 B with coding G6PC promoters containing complete FoxO binding sites (- 1227WT) or mutation FoxO binding sites (- The luciferase fusion constructs transfection FAO liver cells of nucleotide -1227 to+57 1227Mut).It is being not present or is existing Be incubated in the case of 0.1mM cAMP and 1 μM of dexamethasone 16 it is small when after, measure relative luciferase activity.Relatively it is all its It organizes * P<0.001；Compared with untreated Mut**P=0.009 (average value ± S.E.M.).Figure 30 C pass through immunoblot detection From the gland-LacZ and KD-CaMKII CREB or Crtc2 of core of liver of mouse handled and phosphocarnic acid albumen (Np), and detect Glucagon processing comes from WT mouse or Camk2g-/-The p-CREB and beta-actin of the cell extract of the HC of mouse. Figure 30 D detections come from WT mouse or Camk2g from glucagon processing-/-The Crtc2 and phosphocarnic acid of the core of the HC of mouse Albumen (Np).
Figure 31 A-F. are related to Figure 26-FoxO1 phosphorylations.Figure 31 A are to coming from fasting WT mouse and Camk2g-/-Mouse The extract of liver carry out Western blotting with measure phosphoric acid T24-FoxO1, phosphoric acid 253-FoxO1 and phosphoric acid 316-FoxO1 and β- Actin.Figure 31 B are to the WT mouse handled from fasting gland-FoxO1 and Camk2g-/-The extract of the liver of mouse is exempted from Epidemic disease trace is to measure acetylation FoxO1 (Ac-FoxO1) and beta-actin.Average Ac-FoxO:Beta-actin densitometry Ratio seems less than trace；N.s., not significantly.Figure 31 C have the mouse FoxO1 sequences in the identification phosphoric acid site represented with runic； It is shown in the overview of the data of Figure 26,31F and 32 relevant Key residues in lower section in the project list below sequence.Figure 31 D From Camk2g-/-The MS/MS spectrum of No. 4 Phosphorylated Peptides of HC and b, y ion table.Figure 31 E come from Camk2g-/-No. 5 phosphorus of HC It is acidified the MS/MS spectrum of peptide and b, y ion table.Figure 31 F are similar with the experiment in Figure 26 B, with the exception is that L-Foxo1HC is used FLAG-9A-FoxO1 mutant rather than FLAG-7A-Foxo1 transfections (* P=0.006；* P=0.02；Average value ± S.E.M.).
Figure 32 A-E. are related to Figure 26-p38.When Figure 32 A mouse are by arbitrary feeding or small fasting 12.Pass through Western blotting Measure liver phosphoric acid p38, total p38, p-MK2, total MK-2 and beta-actin.Figure 32 B make WT HC be subjected to staying overnight serum exhaustion and connect In the serum free medium with or without SB202190 be incubated with 100nm glucagons 5 it is small when；Or by pancreas hyperglycaemia Element is added to the P38a that gland-LacZ (control) or gland-Cre is handled that uses by oneselffl/flIn the HC of mouse.Measure RNA G6pc and Pck1mRNA(*P<0.05；Average value ± S.E.M.).Figure 32 C are measured from fasting WT mouse and Camk2g-/-The HC's of mouse P-p38, total p38 and beta-actin.Figure 32 D WT mouse 12.5mg kg-1The p38 inhibitor (SB202190) of weight or Injection in intermedium control peritonaeum.After 12 hours, the SB202190 injections of mouse extra dose and overnight fasting.It measures Core FoxO1, phosphocarnic acid albumen, p-MK2, total MK-2 and the beta-actin of liver.Between core FoxO1 and p-MK2 band intensity Association is shown in figure.Figure 32 E make gland-GFP-FoxO1 transduce HC be subjected to stay overnight serum exhaust, and then with When incubation 5 is small in the serum free medium that SB202190 or medium (Veh) compare.FoxO1 is assessed by indirect immunofluorescence Positioning (engineer's scale, 10 μm；*P<0.05；Average value ± S.E.M.).
Figure 33 A-D. calcineurins promote the CRTC2 activation during fasting.Figure 33 A.Ser/Thr phosphatases inhibit Agent (okadaic acid (OA), cyclosporin A (CsA)) is to CRTC2 dephosphorylations and CRE- luciferases (luc) report body (reporter) Influence (the * P of activation<0.001；N=3).Figure 33 B. glucagons (Gcg) are to the wild type (WT) in liver cell and calcium tune god Influence (the * P of dephosphorylation (left side) and active (right side) through phosphoric acid deficient (Δ Calna) CRTC2<0.001；N=3). Figure 33 C. calcineurins A over-express (left side) or strike low (right side) to the CRTC2 dephosphorylations (top) of liver cell, CRE-luc report body activity (intermediate, * P<0.001；N=3) and glucose exports (bottom, * P<0.001；N=3 influence). Figure 33 D. livers calcineurin over-express (left side) or strike low (right side) it is small to fasting 6-8 when mouse in CRE- Influence (the * P of luc activity, gluconeogenesis gene (Pck1, G6pc) expression and blood sugar concentration<0.01；N=5).For this figure and Other figures, data are shown as average value ± s.e.m.
Figure 34 A-D. glucagons stimulate CRTC2 dephosphorylations by activating InsP3 receptors.Figure 34 A. by fluorescence into As come show glucagon (Gcg) in liver cell calcium move influence.Be shown in strike low all 3 InsP3R families into Calcium movement and calcineurin after member activate (* P<0.001；N=3).Figure 34 B. calcium chelating agents (BAPTA) are right CRTC2 dephosphorylations and influence (the * P of CRE-luc activation<0.001；N=3).Figure 34 C.InsP3R exhaust the CRTC2 to liver cell Influence (the * P of dephosphorylation, CRE-luc activity and glucose output<0.001；N=3).Figure 34 D. livers InsP3R strikes low to CRE- Luc activity, blood glucose and influence (the * P of gluconeogenesis gene expression<0.01；N=5).
Figure 35 A-F. glucagons stimulate CRTC2 active by PKA dependence InsP3R phosphorylations.Figure 35 A and B. The Western blotting of precipitation is immunized in the InsP3R1 obtained using phosphoric acid PKA substrates antiserum, shows H89 (A) and at one or two Ala mutation (B) at a (DM) PKA conserved sites (Ser1589, Ser1756) are thin to the liver for being exposed to glucagon (Gcg) The influence of InsP3R1 phosphorylations in born of the same parents.Show wild type InsP3R1 and PKA mutation InsP3R1 to the calcium shifting in response to Gcg Dynamic influence (B) (* P<0.001；N=3).Figure 35 C and D. wild type InsP3R1 or PKA deficiency InsP3R1 (DM) are thin to liver Calcineurin (Calna) activation (C) of born of the same parents and CRTC2 dephosphorylations (C) and CRE-luc activation (D) and glucose are defeated Go out influence (the * P of (D)<0.001；N=3).Figure 35 E. wild types InsP3R1 and PKA deficiency InsP3R1 live to liver CRE-luc Property, fasting blood-glucose and the influence of gluconeogenesis gene expression (G6pc, Pck1) are (compared with wild type * P<0.01；N=5).Figure 35 F. The co-immunoprecipitation of CRTC2 and InsP3R1 in primary hepatocyte.Instruction is to glucagon (100nM；15 minutes) it is sudden and violent Dew.The input level of CRTC2 and InsP3R1 after display core (Nu) and core in (p/Nu) supernatant fraction.
InsP3R activity in Figure 36 A-C. diabetes is raised.Liver CRE- in Figure 36 A. slight of statures mouse and db/db mouse Luc and Calcineurin Activities (* P<0.001；N=5).Figure 36 B. show arbitrary feeding slight of stature mouse, db/db mouse or The relative quantity of InsP3R family members and the Western blotting of phosphorylation in the liver of ob/ob mouse.Instruction is in PKA or AKT sites The InsP3R phosphorylations at place.The InsP3R or calcineurin A of Figure 36 C.RNAi mediations are exhausted in db/db mouse The influence that CRE-luc activity, gluconeogenesis gene expression and hepatic glucose generate, (* P are measured by pyruvic acid tolerance< 0.01；N=5).
Figure 37 A-E. glucagons are lived by calcineurin dependent mechanism come the CRTC2 in cell cultured supernatant Property.Figure 37 A.Ser/Thr inhibitors of phosphatases (okadaic acid (OA), cyclosporin A (CsA)) is to being exposed to glucagon (Gcg) influence of the CRTC2 positioning in Primary mouse liver cell.Engineer's scale, 5 μm.Figure 37 B. calcineurins inhibit Agent (the self inhibitory peptide of calcineurin (CAP), CN585) and PP1/PP2A inhibitor (Calyculin A) To influence (the * P of the CRE- luciferase reporters body activity in primary hepatocyte<0.001；N=3).Figure 37 C and D. wild types CRTC2 and phosphorylation deficiency (S171A, S275A) activity CRTC2 mutant are to CRE-luc activity (C) (* P<0.001；N= 3) handled with the glucagon for influencing Figure 37 D. instructions of CRTC2 cellular localizations.Engineer's scale, 5 μm.Figure 37 E.CsA are to expression Influence (the * P of CRE-luc activity in the cell of wild type CRTC2 or constitutive activity (S171A/S275A) mutation CRTC2< 0.001；N=3).NS, no statistical discrepancy.
Figure 38 A-D. calcineurins adjust the CRTC2 activity in liver cell.Figure 38 A. are to the calcium tune god in CRTC2 Analysis through phosphoric acid enzyme binding site.Different CRTC2 polypeptides and 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor/missing are indicated in schematic diagram.Display is certainly The Diagnosis of Sghistosomiasis of the CRTC2 of the flag marks of the IP recycling of the calcineurin (Calna) of HA marks in HEK293T cells Mark.Wild type CRTC2 polypeptides and mutation CRTC2 polypeptides and calcium tune nerve phosphoric acid in the culture of Figure 38 B. display primary hepatocytes The opposite co-immunoprecipitation combined of enzyme measures.Instruction encoding wild type or calcineurin deficiency (Δ Calna) The CRTC2 of Flag marks adds the cell of the adenovirus infection of the calcineurin A (Calna) of HA marks.It shows from HA- calcium Adjust neural phosphatase IP recycling CRTC2 polypeptides.Wild type (WT) CRTC2 of Figure 38 C. in the liver cell of glucagon It shuttles with the core of calcineurin deficiency (Δ Calna) CRTC2.Engineer's scale, 5 μm.Figure 38 D. use phosphoric acid PKA substrates Antibody (anti-RRXpS/T) measure Calna overexpression (O.E.) or by RNAi mediation strike down into exhaustion it is thin to liver The influence of downstream PKA signal transductions in born of the same parents.
CRTC2 activity in Figure 39 A-B. calcineurins regulation and control liver.Calcineurin (Calna) excessive table Up to the exhaustion (B) that (A) or RNAi are mediated in the liver of fasted mice fasting inductivity CREB target genes (Nr4a1, Nr4a2, Pgc1 α) mRNA amounts influence (* P<0.01；N=5).Display is over-expressed compared to control from calcineurin Or strike the Western blotting of the opposite calcineurin albumen quality in the liver of low mouse.
Figure 40 A-C. calcineurins and InsP3R regulate and control gluconeogenesis by adjusting CRTC2 activity.Figure 40 A-B. The fasting of calcineurin or InsP3R1 that the Crtc2RNAi of adenovirus coding is encoded to expressing adenovirus as indicated The influence of blood sugar concentration (A) and liver gene expression (B) in mouse.N=4.NS, no statistical discrepancy.Figure 40 C. display figures A and B The Western blotting of the orgotein quality of CRTC2, calcineurin (Calna) and InsP3R1 in the mouse of middle characterization.
Calcium movement in Figure 41 A-C. glucagon cell cultured supernatants.The pancreas that Figure 41 A and B. is measured by fluorescence imaging Influence (the * P that glucagons (Gcg) and PKA inhibitor H89 move the calcium in primary hepatocyte<0.001；N=3).Figure 41 C. By to phosphoric acid PKA substrates antiserum to come from be exposed to glucagon (Gcg) primary hepatocyte lysate system The InsP3R1 peptides that the MS of standby immune precipitation is analyzed and identified.
Figure 42 A-E.InsP3 receptors are in response to needed for the CRTC2 activation of cAMP agonists.The smooth Spongistatin of Figure 42 A, B. C (Xc) is to the calcium movement being exposed in the primary hepatocyte of forskolin (FSK) and CRTC2 dephosphorylations (A) and to glucose -6- Influence (the * P of the mRNA amounts (B) of phosphatase (G6pc) and PEPCK (Pck1)<0.001；N=3).Figure 42 C-E. glucagons And calcineurin activation mobile to the calcium in the primary hepatocyte from InsP3R2 knock-out mices and control littermate (C)(*P<0.001；N=4) and CRE-luc report bodies activate and glucose production (D) (* P<0.001；) and CRTC2 n=4 The influence of dephosphorylation (E).Figure 42 E. right bars figures are shown that exposure to the wild type hepatocytes of glucagon and InsP3R2 strikes Except the opposite CRTC2 dephosphorylations in liver cell, (* P are measured by densitometry art<0.01；N=3).
Figure 43 A-E.InsP3R adjust hepatic gluconeogenic.All 3 InsP3R family members of Figure 43 A.RNAi mediations exhaust Influence (the * P activated to the calcineurin in fasting liver<0.001；N=5).Figure 43 B and C. livers InsP3R strikes low to small The mRNA amounts (B) of fasting inductivity CREB target genes in the liver of mouse and influence (the * P to InsP3R albumen qualities (C)< 0.01；N=5).Measurement is tested by pyruvic acid tolerance in Figure 43 D and E. wild-type mice and InsP3R2 knock-out mices Hepatic gluconeogenic (D) (* P<0.02；**P<0.01；) and gluconeogenesis gene expression (E) (* P n=4<0.01；N=4).Western blotting Show the InsP3R2 albumen qualities in the liver extract from control mice and InsP3R2 knock-out mices.
Figure 44 A-D. glucagons adjust InsP3R activity by the phosphorylation that PKA is mediated.Figure 44 A. use phosphoric acid The display glucagon (Gcg) that the immune precipitation of InsP3R of the PKA substrates antiserum to being prepared from primary hepatocyte obtains is right The Western blotting of the influence of InsP3R phosphorylations.Figure 44 B. show liver when fasting 6-8 is small or when feeding 2 is small again in Mouse Liver The Western blotting of InsP3R phosphorylations at PKA or AKT sites.Figure 44 C and D. wild type InsP3R1 and PKA deficiencies (DM) InsP3R1 activates (C, * P to the calcineurin (Calna) in fasting liver<0.001, n=5) and CREB target genes (D, * P<0.01, n=5) influence.Right side, the gland of display encoding wild type InsP3R1 or PKA deficiency (DM) InsP3R1 The Western blotting of opposite InsP3R albumen qualities in the liver of the mouse of virus injection.
The association of InsP3R and CRTC2 in Figure 45 A-I. liver cells.Precipitation recycling is immunized from anti-CRTC2 in Figure 45 A. InsP3R1 peptides (are analyzed) according to MS.Figure 45 B. show the IP recycling of the InsP3R1 from the HA marks in HEK293T cells The co-immunoprecipitation of the amount of the CRTC2 of Flag marks measures.Figure 45 C and D. is to being CRTC2 in CRTC2 (C) and InsP3R1 (D): The deletion analysis in the region needed for InsP3R1 interactions.In each schematic diagram instruction interaction competence CRTC2 and InsP3R1 polypeptides (+).Figure 45 E.InsP3R exhaust to be exposed in the liver cell of glucagon (Gcg) with ER enrichment The influence of the relevant CRTC2 albumen qualities of high density microsome (HDM) and cytosol (Cyto) and core (Nucl) part.Instruction The relative quantity of ER positioning (GRP78), cytosol (tubulin) and core (CREB) protein in each several part.Figure 45 F.InsP3R Exhaust the influence positioned to the CRTC2 in liver cell.Engineer's scale, 5 μm.Figure 45 G-I. wild types, InsP3R deficiencies (Δ CBD, 51-692aa) and the cellular localization of myristoylation CRTC2 mutant polypeptides (G), phosphorylation state (H) and activity (I) (* P< 0.001；N=3).Engineer's scale, 5 μm.
The InsP3R phosphorylations that Figure 46 A-D. insulin is mediated by AKT are active to lower CRTC2.On the left of Figure 46 A., make The display insulin (INS) obtained with the immune precipitation of InsP3R of the phosphoric acid AKT substrates antiserum to being prepared from primary hepatocyte The Western blotting of influence to InsP3R phosphorylations.Bottom right, insulin (INS) is to wild type InsP3R1 and in shared AKT Contain the influence of the phosphorylation of the mutation InsP3R1 of alanine substitution at phosphorylation site (Ser2682).Figure 46 B. insulin pair The glucagon (Gcg) expressed in the primary hepatocyte of wild type InsP3R1 or AKT deficiency (S2682A) InsP3R1 lures The calcium movement led and influence (the * P of calcineurin (Calna) activation<0.001；N=3).Figure 46 C. wild types InsP3R1 and AKT deficiencies (S2862A) InsP3R1 reports that body is lived to CRTC2 dephosphorylations (top), the CRE-luc of liver cell Change influence (the * P of (bottom left) and glucose output (bottom right)<0.001；N=3).Figure 46 D. wild types InsP3R1 and CRE-luc activity, blood glucose and CREB target genes of AKT deficiencies (S2682A) InsP3R1 to fasting or again in feeding mouse Express influence (the * P of (G6pc, Pck1, Nr4a1, Nr4a2, Pgc1 α)<0.01；N=5).Bottom right, display coding are wild Opposite InsP3R protein in the liver of the mouse of the adenoviral injection of type InsP3R1 or AKT deficiency (S2682A) InsP3R1 Western blotting (the * P of amount<0.01；N=5).
Liver cAMP signal transductions and Calcineurin Activities in Figure 47 A-C. obesity increase.Figure 47 A and B. is thin In mouse, db/db mouse and ob/ob mouse liver CRE-luc report body activity and Calcineurin Activities (A) and CAMP contents (B) (* P<0.001；N=5).In livers of Figure 47 C. with the mouse of Insp3r or Calna RNAi adenoviral injections The Western blotting of InsP3R and calcineurin (Calna) albumen quality.
Figure 48 fasting and feeding approach regulate and control CRTC2 dependence gluconeogenesises by the antagonism to InsP3R activity. Fasting signal transduction activates InsP3R by PKA dependences phosphorylation, so as to cause calcineurin (Calna) activity Increase with subsequent CRTC2 dephosphorylations.On the contrary, feeding is active by AKT dependence inhibition of phosphorylation InsP3R, so as to block calcium tune Neural phosphatase dependence CRTC2 dephosphorylations.
In the G6Pase/PEPCK of forskolin/glucagon inductions of Figure 49 A-S.IP3R in the liver cell of culture Effect and the effect in fasting blood-glucose in vivo.The G6Pase and PEPCK of Figure 49 A, B.FSK induction are Ca-dependents.Figure (FAO rat hepatocytes cells needed for the G6Pase and PEPCK that 49C-D.IP3R activity induces for forskolin and glucagon System).Figure 49 E-F.IP3R activity is needed for the G6Pase and PEPCK of forskolin induction (primary hepatocytes).Figure 49 G- The G6Pase and PEPCK (primary hepatocytes) of H.IP3R1 antisense oligonucleotides containment forskolin induction.Figure 49 I. forskolins are lived Change the IP3R activity in FAO liver cells (the calcium release of inositol/ATP inductions measures).Figure 49 J.PKA inhibitor H89 contains hair larynx The IP3R activation of element induction.Figure 49 K-L.IP3R activity is needed for the PGC1 α of forskolin induction.Figure 49 M-N. calcium tune nerve phosphoric acid The G6Pase and PEPCK of enzyme inhibition peptide containment forskolin induction.Figure 49 O.IP3R activity is neural for the calcium tune of forskolin induction Needed for phosphoric acid enzyme activation.Figure 49 P-Q.IP3R activity is needed for the TORC2 dephosphorylation and nuclear location of forskolin induction.Figure 49R.TORC2 transduction moieties recover the G6Pase of the forskolin induction in the liver cell that IP3R inhibits.The smooth Spongistatin C of Figure 49 S. Processing reduces the fasting glucose level in C57BL/6J mouse.
Effects of Figure 50 A-M.IP3R in the hyperglycaemia and gluconeogenesis of obesity/insulin resistance induction.Figure 50 A. light It slips Spongistatin C processing ob/ob mouse and inhibits the evidence that the IP3R of the IICR in the peritoneal macrophages of fresh separated is measured.Figure 50B-E. inhibits the fasting blood glucose level in IP3R reduction ob/ob mouse.Figure 50 F. inhibit the liver in IP3R reductions ob/ob PEPCK, G6Pase and PGC1 alpha expression.Figure 50 G-I. inhibit IP3R reductions stress be with the palm fibre of the model of insulin resistance as ER PEPCK, G6Pase and PGC1 alpha expression in the liver cell of palmitic acid hydrochlorate processing.Figure 50 J-K., which inhibit IP3R, to be reduced as insulin G6Pase and PEPCK expression in the liver cell that the Akt of the model of resistance inhibits.Figure 50 L.IP3R activity is forskolin induction Needed for TORC2 dephosphorylations.Figure 50 M.IP3R activation is needed for the reservation of TORC2 cores.
Figure 51 show the figure of IP3R approach.
Figure 52 .CA-CaMKII do not increase the core FoxO1 in the liver cell of p38 shortages.It is transduceed with gland-LacZ or gland-Cre From p38fl/flThe primary hepatocyte (HC) of mouse.After 12 hours, half cell with gland-CA-CaMKII transduce and in addition Half is transduceed with gland-LacZ.Cell is incubated overnight in serum exhausts culture medium and then incubation 5 is small in serum free medium When.The FoxO1 of nuclear extract and phosphocarnic acid albumen (Np) are measured by Western blotting.
Figure 53 A-B.MK-2 regulate and control liver FoxO1 Subcellular Localizations and liver G6Pc and Pck1.Figure 53 A. make gland-GFP-FoxO1 The HC of transduction is subjected to staying overnight serum exhaustion, and then in the serum-free with 2uM MK-2 inhibitor or medium (Veh) control It is incubated 30 minutes in culture medium.By indirect immunofluorescence assess FoxO1 positioning (engineer's scale, 10 μm；*P<0.005；Average value ±S.E.M.).Figure 53 B make WT HC be subjected to staying overnight serum exhaustion and then in the serum free medium with or without MK-2 inhibitor In with 100nm glucagons be incubated with 5 it is small when.Measure G6pc and Pck1mRNA (the * P of RNA<0.005；Average value ± S.E.M.).MK-2 inhibitor comes from Calbiochem (catalog number (Cat.No.) 475864)：2- (2- quinoline -3- yl pyridines -4- bases) -1,5, 6,7- tetrahydrochysene -4H- pyrrolo-es-[3,2-c] pyridine -4- ketone.
Figure 54 .p38 lack the UPR activation of containment tunicamycin induction.By tunicamycin be added to use by oneself gland-LacZ controls or The p38 of gland-Cre transductionsfl/flIn the HC of mouse.To lysate carry out Western blotting with measure p-PERK, PERK, CHOP, P58IPK and actin.
Figure 55 .p38 lack the UPR activation of containment palmitate induction.Palmitate is added to-LacZ pairs of gland of using by oneself According to or gland-Cre transduction p38fl/flIn the HC of mouse.Western blotting is carried out to lysate to measure p-PERK, CHOP and flesh Filamentous actin.
Acute insulin-induced p-AKT in Figure 56 .HC is lacked by p38 to be enhanced.As in Figure 55, the exception It is that HC is handled 5 minutes with 100nM insulin.Western blotting is carried out to lysate to measure p-Akt, Akt and actin.
When Figure 57 A-E. Figure 57 A. mouse are by arbitrary feeding or small fasting 12.By Western blotting measure liver phosphoric acid p38, always P38, p-MK2, total MK-2 and beta-actin.Figure 57 B make WT HC be subjected to stay overnight serum exhaust and then with or without Be incubated in the serum free medium of SB202190 with 100nm glucagons 5 it is small when；Or glucagon is added to come In the HC of the P38afl/fl mouse of personal gland-LacZ (control) or gland-Cre processing.Measure G6pc and Pck1mRNA (the * P of RNA <0.05；Average value ± S.E.M.).Figure 57 C. measure the p-p38, total of the HC from fasting WT mouse and Camk2g-/- mouse P38 and beta-actin.The p38 inhibitor (SB202190) or medium pair of Figure 57 D.WT mouse 12.5mg kg-1 weight According to being injected in peritonaeum.After 12 hours, the SB202190 injections of mouse extra dose and overnight fasting.Measure the core of liver FoxO1, phosphocarnic acid albumen, p-MK2, total MK-2 and beta-actin.Associating between core FoxO1 and p-MK2 band intensities shows It is shown in figure.The HC that Figure 57 E. make gland-GFP-FoxO1 transduce, which is subjected to staying overnight serum, to be exhausted, and then with SB202190 or When incubation 5 is small in the serum free medium of medium (Veh) control.FoxO1 is assessed by indirect immunofluorescence and positions (ratio Ruler, 10 μm；*P<0.05；Average value ± S.E.M.).
Figure 58 .p38 lack the UPR activation for reducing tunicamycin induction.It is used in MOI for 5 times and compares gland-LacZ (Ad-LacZ) Or to lack the gland-Cre (Ad-Cre) of p38 transductions from p 38 alphaflox/floxThe primary hepatocyte of mouse.After when 36 is small, Cell with 0.5ug/ml both tunicamycin treatments 4 it is small when.Then to lysate carry out Western blotting with measure p-Perk, CHOP, Trb3, p58IPK and actin.
Figure 59 .p38 lack the insulin resistance of passivation palmitate induction.In MOI gland-LacZ (Ad-LacZ) is used for 5 times Or gland-Cre (Ad-Cre) transductions are from p38fl/flThe primary hepatocyte of mouse.At 24 hours later, cell is at palmitate Reason is overnight.After when incubation 5 is small in serum free medium together with palmitate, cell handles 5 points with 100nM insulin Clock.Then Western blotting is carried out to lysate to measure p-Akt, Akt, actin, p-perk, CHOP and Trb3.
Figure 60 .CA-CaMKII do not increase the core FoxO1 in the liver cell of p38 shortages：CaMKII and p38 is in identical letter Evidence in number pathway.It is come from MOI for 5 times with gland-LacZ (Ad-LacZ) or gland-Cre (Ad-Cre) overnight transduction p38fl/flThe primary hepatocyte of mouse is then transduceed in MOI for 1 time with gland-LacZ or gland-CA-CaMKII (CA).When 12 is small Afterwards, make cell be subjected to stay overnight serum exhaust and then in serum free medium be incubated 4 it is small when.Core is measured by Western blotting The FoxO1 and phosphocarnic acid albumen of extract (core loads control).
Pck1 and the G6Pc induction of Figure 61 dominant negatives (DN)-MK2 containment forskolin inductions.With expression LacZ or DN- Primary hepatocyte of the adenoviral vector transduction of MK2 from WT mouse.Then cell is made to be subjected to staying overnight serum exhaustion and is then existed Be incubated in serum free medium with 2 μM of forskolins 5 it is small when.The G6pc and Pck1mRNA of RNA is measured by RT-qPCR.*, P <0.0001
It is horizontal that Figure 62 .DN-MK2 reduce core FoxO1.In MOI gland-LacZ (LacZ) or gland-DN-MK2 (DN- is used for 1 time MK2) primary hepatocyte of the transduction from WT mouse.At 24 hours later, cell is made to be subjected to staying overnight serum exhaustion and then in nothing When incubation 4 is small in blood serum medium.The FoxO1 of nuclear extract and phosphocarnic acid albumen (core loading pair are measured by Western blotting According to).
Figure 63 constitutive activities (CA)-CaMKII does not increase the core FoxO1 in the liver cell of MK-2 inhibition：CaMKII and MK2 is in the evidence in identical signal transduction path.In MOI gland-LacZ (LacZ) or gland-DN-MK2 (DN-MK2) is used for 1 time Primary hepatocyte of the overnight transduction from WT mouse then uses gland-LacZ or gland-CA-CaMKII (CA- in MOI for 1 time CaMKII) transduce.After 12 hours, being subjected to cell, overnight serum is exhausted and then incubation 4 is small in serum free medium When.The FoxO1 of nuclear extract and phosphocarnic acid albumen (core loads control) are measured by Western blotting.
Figure 64 livers MK-2 inhibition does not change weight or food intake.With 1x10^9pfu containing control LacZ (n=5) or The 10 week old ob/ob mouse of adenoviral injection of DN-MK-2 (n=5)
Figure 65 livers MK-2 inhibits to improve the hyperglycaemia in obesity mice.Contain control LacZ (n=5) with 1x10^9pfu Or the 10 week old ob/ob mouse of adenoviral injection of DN-MK-2 (n=5).On day 3 when fasting 6 is small after blood glucose water It is flat；*=p<0.05
Figure 66 livers MK-2 inhibits to improve the hyperglycaemia (absolute value) in obesity mice.Glucose tolerance was carried out at the 5th day Test *=p (for each group, n=5) (in 0.5g/kg peritonaeums)<0.05, * *=p<0.01, * * *=p<0.005
Figure 67 livers MK-2 inhibits to improve the hyperglycaemia in obesity mice (compared with time=0).Grape was carried out at the 5th day Sugared tolerance test *=p (for each group, n=5) (in 0.5g/kg peritonaeums)<0.05, * *=p<0.01, * * *=p<0.005
Figure 68 livers MK-2 inhibits to improve the hyperinsulinemia of obesity mice.Contain control LacZ (n with 1x10^9pfu =5) or 10 week old ob/ob mouse of the adenoviral injection of DN-MK-2 (n=5).The 8th day when fasting 6 is small after serum pancreas Island element is horizontal；*=p<0.05
Pck1 and the G6Pc induction of forskolin induction in Figure 69 .MK-2 inhibitor containment primary hepatocyte.Make from WT The primary hepatocyte of mouse is subjected to staying overnight serum exhaustion and then uses 300nM MK-2 inhibitor (compound 28) or medium pre- Handle 1 it is small when.Cell then in the serum free medium with or without inhibitor with 2 μM of forskolins handle 4 it is small when.Pass through RT- QPCR measures the G6pc and Pck1mRNA of RNA.*, P<0.0001.
Figure 70-76 is shown in the MK-2 inhibitor research (the compound 28* from Huang etc.) carried out in obesity mice. By being injected in peritonaeum (with 150ul total volumes) daily to ob/ob mouse application-MK-2 inhibitor (10 μ g/ mouse；0.2mg/ ) or intermedium control 1 time kg.Referring to Huang X, Zhu X, Chen X, Zhou W, Xiao D, Degrado S, Aslanian R, Fossetta J, Lundell D, Tian F, Trivedi P, Palani A.Bioorg Med Chem Lett.2012 1 The moon 1；22(1):65-70,“A three-step protocol for lead optimization:quick identification of key conformational features and functional groups in the SAR studies of non-ATP competitive MK2(MAPKAPK2)inhibitors”.Department of Medicinal Chemistry,Merck Research Laboratories,2015Galloping Hill Road, Kenilworth,NJ07033,USA。
Figure 70 shows that MK-2 inhibitor does not change weight.It is applied daily to 10 week old ob/ob mouse by being injected in peritonaeum MK-2 inhibitor 1 time (150ul total volumes).
Figure 71 shows that MK-2 inhibitor does not change food intake.By being injected in peritonaeum daily to 10 week old ob/ob mouse Using MK-2 inhibitor 1 time (150ul total volumes).
Figure 72 shows that MK-2 inhibitor reduces fasting blood-glucose.It is applied daily to 10 week old ob/ob mouse by being injected in peritonaeum With MK-2 inhibitor 1 time (150ul total volumes).The fasting blood glucose level when the 6 of the 5th day is small.*, p<0.05
Figure 73 shows that MK-2 inhibitor improves the hyperglycaemia in obesity mice.By being injected in peritonaeum daily to 10 week old Ob/ob mouse apply MK-2 inhibitor 1 time (150ul total volumes).Glucose tolerance test (0.5g/kg abdomens were carried out at the 7th day In film).*, p<0.05
Figure 74 shows that MK-2 inhibitor improves the insulin resistance in obesity mice.By being injected in peritonaeum daily to 10 weeks Age ob/ob mouse applies MK-2 inhibitor 1 time (150ul total volumes).Insulin resistance test (1.5IU/ was carried out at the 12nd day kg).*, p<0.05
Figure 75 shows that MK-2 inhibitor reduces Fasting insulin level.By being injected in peritonaeum daily to 10 week old ob/ob Mouse applies MK-2 inhibitor 1 time (150ul total volumes).The Fasting insulin level when the 6 of the 10th day is small.*, p<0.05
Figure 76 shows that MK-2 inhibitor does not change total WBC.It is applied daily to 10 week old ob/ob mouse by being injected in peritonaeum MK-2 inhibitor 1 time (150ul total volumes).In total WBC count levels of the 18th day.
The FoxO1 phosphorylation in vitro that Figure 77 are reached by p38.1ug WT GST-FoxO1 or GST control is made to be activated with 25ug P38 (Upstate Biotechnology) is incubated 30 minutes in 15ul kinase buffer liquids at 30 DEG C together, the buffer solution Contain 25mMTris-HCl (pH7.5), 5mM β-glycerophosphate, 2mM dithiothreitol (DTT)s (DTT), 0.1mM Na3VO4,10mM MgCl2 and 2.5 μ Ci [γ -32P] ATP.It is observed by SDS-PAGE separating samples and by autoradiograph, with laggard Row immunoblotting assay.
The FoxO1 phosphorylation in vitro that Figure 78 are reached by MK-2.Make 1ug WTGST-FoxO1, GST (control) or GST- Hsp25 is with or without 25ug activation MK-2 (Upstate Biotechnology) together in 15ul kinase buffer liquids at 30 DEG C Lower to be incubated 30 minutes, the buffer solution contains 25mM Tris-HCl (pH7.5), 5mM β-glycerophosphate, 2mM dithiothreitol (DTT)s (DTT), 0.1mM Na3VO4,10mM MgCl2 and 2.5 μ Ci [γ -32P] ATP.By SDS-PAGE separating samples and by putting It penetrates autography to be observed, then carries out immunoblotting assay.
Figure 79 dominant-negatives MK2 reduces the liver G6pc in ob/ob mouse.Use 1x109Pfu's contains control LacZ (n= Or 9 week old ob/ob mouse of the adenoviral injection of DN-MK2 (n=5) 5).8 days analysis liver G6pc mRNA after injection.(*p< 0.05)。
Figure 80 .MK-2 inhibitor reduces the FoxO1 target genes in the liver of ob/ob mouse.By being injected in peritonaeum, per daily MK-2 inhibitor (0.2mg/kg) or intermedium control handle 10 week old ob/ob mouse 1 time, continue 20 days.Divided by qRT-PCR Analyse liver G6pc, Pck1 and Igfbp-1mRNA.(*p<0.05, * * p<0.05).The MK-2 inhibitor used in Figure 80-85 is following Described in compound 28：Huang X,Zhu X,Chen X,Zhou W,Xiao D,Degrado S,Aslanian R, Fossetta J, Lundell D, Tian F, Trivedi P, Palani A.Bioorg Med Chem Lett.2012 January 1 day；22(1):65-70,“Athree-step protocol for lead optimization:quick identification of key conformational features and functional groups in the SAR studies of non-ATP competitive MK2(MAPKAPK2)inhibitors”.Department of Medicinal Chemistry,Merck Research Laboratories,2015Galloping Hill Road, Kenilworth,NJ07033,USA。
Figure 81：MK-2 inhibitor improves the liver insulin signal transduction in ob/ob mouse.By being injected in peritonaeum, daily 10 week old ob/ob mouse are handled with MK-2 inhibitor (0.2mg/kg) or intermedium control 1 time, continue 20 days.When fasting 6 is small Afterwards, insulin (2IU/kg) is injected in mouse by portal vein, continues 3 minutes.Pass through western blot analysis liver phosphorus Sour Akt (serine 473) and total Akt are horizontal.The densitometry quantitative display of immunoblotting data is in figure.(*p<0.05).
Figure 82 .MK-2 inhibitor reduces the Chop and Trib3mRNA in the liver of ob/ob mouse.As in Figure 80, example Outer part is that analysis liver Chop and Trb3mRNA are horizontal.(*p<0.05).
Figure 83 .MK-2 inhibitor reduces the FA synthesis mRNA and Tnfa mRNA in the liver of ob/ob mouse.As in Figure 80 Equally, with the exception is that analysis liver Fas, Scd1, Srebp1, Tnfa mRNA level in-site.(*p<0.05).
It is additive that the FBG of Figure 84 .MK-2 inhibitor mediation, which is reduced with melbine,.One time a day with intermedium control, At the injection of independent MK-2 inhibitor (0.2mg/kg), independent melbine (250mg/kg) or MK-2 inhibitor and melbine 10 week old ob/ob mouse are managed, continue 5 days.Show 6 it is small when fasting blood glucose level.(*p<0.05).
It is additive that the liver G6pc of Figure 85 .MK-2 inhibitor mediation, which is reduced with melbine,.As in Figure 84, example Outer part is to analyze liver G6Pc mRNA by qRT-PCR.(*p<0.05).
Figure 86 .p38 lack the UPR activation of containment tunicamycin induction and Trb3 up-regulations.MOI for 10 time with expression LacZ or Primary mouse liver cell of the adenoviral vector transduction of Cre from p38floxed mouse.When after transfection 36 is small, cell is used When tunicamycin (1 μ g/ml) processing 4 is small.Phosphoric acid Perk, Perk, Chop, β-flesh of cell lysates are measured by Western blotting Filamentous actin and Trb3.
It is horizontal that Figure 87 .p38 lack containment Trb3mRNA.It is that 10 times adenovirus vectors with expression LacZ or Cre turn in MOI Lead the Primary mouse liver cell from p38floxed mouse.When after transfection 24 is small, cell is at palmitate (300 μm) Manage 18 it is small when.Trb3mRNA is measured by qRT-PCR.(, P<0.0005；*, P<0.0001).
Acute insulin-induced p-AKT enhancings in Figure 88 .p38-/- liver cell are drawn by being transduceed with Ad-Trb3 It rescues.It it is 10 times thin with Primary mouse liver of the adenoviral vector transduction from p38floxed mouse for expressing LacZ or Cre in MOI Born of the same parents.After being incubated overnight, the cell gland-Trb3 of half gland-Cre processing transduces and remaining cell receives gland-LacZ.12 After hour, cell with (300 μm) of palmitate processing 14 it is small when, when then processing 5 is small in serum free medium.In no blood After when incubation 5 is small in clear culture medium, cell is stimulated 5 minutes with insulin (100nm).Then cell is measured by Western blotting The phosphoric acid Akt (serine 473) of lysate, Akt, Trb3 and beta-actin.
Detailed description of the invention
Patent and scientific literature mentioned in this article determine can be the knowledge used in those skilled in the art.It is cited herein Give patent, application and other announcements be hereby incorporated herein by accordingly, the degree of the reference is just as clearly Ground and individually instruction are each herein incorporated by reference general.
It will such as show and be apparent from for those of ordinary skill in the art, any method as described herein or composition all can be on these Any other method or composition described in text are performed.
Definition and abbreviation
Unless otherwise context is in addition clearly stipulate that singulative " one (a) ", " a kind of (an) " and " described " include answering It is several to refer to object.
When being used together in claim and/or specification with term "comprising", using wording " one (a) " or " a kind of (an) " can mean "one", but it is also complied with " one or more ", " at least one " and " one or more " Meaning.
Term " about " herein for refer to it is approximate, in ... left and right, roughly or about.When term " about " is together with numerical value model It encloses when being used together, it is by extending above border and modifying that scope less than the numerical value.In general, term " about " it is used to be higher than herein and changes to modify numerical value less than described value 20%.
As used herein, abbreviation " CaMKII " refers to enzyme --- calcium Calmodulin depedent kinase II includes its hypotype With their splice variant any one.The nucleotide sequence of the gene of the different subtype of CaMKII, bag are encoded as is generally known in the art It includes but is not limited to the nucleotide sequence of the open reading frame of gene.The gene of the different subtype of encoding human CaMKII as is generally known in the art Nucleotide sequence, include but not limited to gene open reading frame nucleotide sequence.CaMKII polypeptides and egg as is generally known in the art The amino acid sequence of white matter includes but not limited to the amino acid sequence of people's CaMKII peptide and proteins.It transcript variant and cuts The sequence for connecing variant be also known in the art (see, for example, Couchonnal and Anderson, 2008, with reference Mode is integrally incorporated herein).The accession number of the nucleotide sequence of house mouse (mus musculus) CaMKII- γ is NM_178597 And the accession number of the nucleotide sequence of people CaMKII- γ is NM_172171.2.The amino acid sequence of house mouse CaMKII- γ is stepped on Record number is that the accession number of NP_848712.2 and the amino acid sequence of people CaMKII- γ is NP_751911.1.
As used herein, abbreviation " IP3R " refers to Isosorbide-5-Nitrae, 5- inositol triphosphate receptors, including its hypotype and they cut Connect any one of variant.In IP3R families, several hypotype has been identified and characterized.Abbreviation " IP3R1 " refers to I types IP3R Asias Type, abbreviation " IP3R2 " refers to II type IP3R hypotypes, and abbreviation " IP3R3 " refers to type III IP3R hypotypes.It compiles as is generally known in the art The nucleotide sequence of the gene of the different subtype of code IP3R includes but not limited to the nucleotide sequence of the open reading frame of gene.Ability The nucleotide sequence of the gene of the different subtype of known coded people IP3R in domain includes but not limited to the core of the open reading frame of gene Acid sequence.The amino acid sequence of IP3R peptide and proteins as is generally known in the art includes but not limited to the people of different IP3R hypotypes The amino acid sequence of IP3R peptide and proteins.The accession number of the nucleotide sequence of house mouse IP3R1 is NM_010585.5 and people The accession number of the nucleotide sequence of IP3R1 is NM_001099952.2.The accession number of the amino acid sequence of house mouse IP3R1 is NP_ 034715.3 and the accession number of the amino acid sequence of people IP3R1 be NP_001093422.2.For the other information in relation to IP3R, Referring to Wehrens etc., 2005, Annu Rev Physiol., 67:69-98.“Intracellular calcium release and cardiac disease”；Referring also to Patterson etc., 2004, Annu Rev Biochem.73:437-65. " Inositol 1,4,5-trisphosphate receptors as signal integrators. " and Volpe etc., 1990,Am J Physiol.；258(6Pt1):C1086-91.“Regulation of inositol 1,4,5- trisphosphate-induced Ca2+release.II.Effect of cAMP-dependent protein Kinase. ", whole is hereby incorporated herein by.
As used herein, abbreviation " p38 " refers to protein (MAP) kinases of any p38 mitogens (mitogen) activation. Identified several p38MAP kinases, including p38- α (also referred to as MAPK14), p38- β (also referred to as MAPK11), p38- γ ( Referred to as MAPK12/ERK6) and p38- δ (also referred to as MAPK13/SAPK4).The nucleic acid sequence of the gene of p38 is encoded as is generally known in the art Row include but not limited to the nucleotide sequence of the open reading frame of gene.The nucleic acid sequence of the gene of encoding human p38 as is generally known in the art Row include but not limited to the nucleotide sequence of the open reading frame of gene.The amino acid of p38 peptide and proteins as is generally known in the art Sequence includes but not limited to the amino acid sequence of people's p38 peptide and proteins.The nucleotide sequence of house mouse p38- α (MAPK14) Accession number be that the accession number of NM_011951.3 and the nucleotide sequence of people p38- α (MAPK14) is NM_001315.2.House mouse The accession number of the amino acid sequence of p38- α (MAPK14) is the amino acid sequence of NP_036081.1 and people p38- α (MAPK14) Accession number is NP_001306.1.For the other information in relation to p38, referring to Marber etc., 2011, J Mol Cell Cardiol.；51(4):485-90“The p38mitogen-activated protein kinase pathway--a potential target for intervention in infarction,hypertrophy,and heart failure.”；Referring also to Kostenko etc., 2011, World J Biol Chem.26；2(5):73-89. “Physiological roles of mitogen-activated-protein-kinase-activated p38- Regulated/activated protein kinase. " and Cuadrado etc., 2010, Biochem J.；429(3):403- " 17. Mechanisms and functions of p38MAPK signalling. ", whole is herein incorporated by reference this Text.
As used herein, abbreviation " MK2 " refers to the kinases MK2 of p38 activation, and the protein of also referred to as map kinase activation swashs Enzyme 2 (or MAPKAPK2).The nucleotide sequence of the gene of MK2 is encoded as is generally known in the art, and the opening for including but not limited to gene is read The nucleotide sequence of frame.The nucleotide sequence of the gene of encoding human MK2 as is generally known in the art, the opening for including but not limited to gene are read The nucleotide sequence of frame.The amino acid sequence of MK2 peptide and proteins as is generally known in the art includes but not limited to people's MK2 polypeptides With the amino acid sequence of protein.The accession number of the nucleotide sequence of house mouse MK2 is the nucleotide sequence of NM_008551 and people MK2 Accession number be NM_004759.4.The accession number of the amino acid sequence of house mouse MK2 is the amino of NP_032577.1 and people MK2 The accession number of acid sequence is NP_004750.1.
As used herein, abbreviation " MK3 " refers to the kinases MK3 of p38 activation, and the protein of also referred to as map kinase activation swashs Enzyme 3 (or MAPKAPK3).The nucleotide sequence of the gene of MK3 is encoded as is generally known in the art, and the opening for including but not limited to gene is read The nucleotide sequence of frame.The nucleotide sequence of the gene of encoding human MK3 as is generally known in the art, the opening for including but not limited to gene are read The nucleotide sequence of frame.The amino acid sequence of MK3 peptide and proteins as is generally known in the art includes but not limited to people's MK3 polypeptides With the amino acid sequence of protein.Abbreviation " MK2/3 " refers to MK2 or MK3 or both MK2 and MK3.The nucleic acid sequence of house mouse MK3 The accession number of row is that the accession number of NM_178907.3 and the nucleotide sequence of people MK3 is NM_001243926.1.House mouse MK3's The accession number of amino acid sequence is that the accession number of NP_849238 and the amino acid sequence of people MK3 is NP_004626.1.For having The other information of MK2 and MK3 is closed, referring to Gaestel etc., 2006, Nat Rev Mol Cell Biol.7 (2):120-30. “MAPKAP kinases-MKs-two's company,three's a crowd.”；Referring also to Shiryaev etc., 2010, Cell Signal.；22(8):1185-92.“Mitogen-activated protein kinase p38and MK2, MK3and MK5:ménage à trois or ménageàquatre" and Kotlyarov etc., 2002, Biochem Soc Trans.；30(Pt6):959-63.“Is MK2(mitogen-activated protein kinase-activated protein kinase2)the key for understanding post-transcriptional regulation of gene expression", whole is hereby incorporated herein by.
As used herein, term " calcineurin " refers to that the catalysis of protein phosphatase calcineurin is sub- Regulation and control subunit of unit or protein phosphatase calcineurin or both, including their hypotype and their montage Any one of variant.The nucleic acid sequence of the gene of the different subtype of the subunit of calcineurin is encoded as is generally known in the art Row include but not limited to the nucleotide sequence of the open reading frame of gene.Encoding human calcineurin as is generally known in the art The nucleotide sequence of the gene of the different subtype of subunit includes but not limited to the nucleotide sequence of the open reading frame of gene.Ability It is more to include but not limited to people's calcineurin for the amino acid sequence of known calcineurin peptide and protein in domain The amino acid sequence of peptide and protein.The accession number of the nucleotide sequence of house mouse calcineurin subunit B is NM_ 024459.2 and the accession number of the nucleotide sequence of people's calcineurin catalytic subunit be NM_000944.4.House mouse calcium The accession number for adjusting the amino acid sequence of neural phosphatase subunit B is NP_077779.2 and the catalysis of people's calcineurin is sub- The accession number of the amino acid sequence of unit is NP_000935.1.For the other information in relation to calcineurin, referring to Wilkins etc., 2004, Biochem Biophys Res Commun.1；322(4):1178-91.“Calcium- calcineurin signaling in the regulation of cardiac hypertrophy.”；Referring also to Periasamy,2002,J Mol Cell Cardiol.34(3):259-62.“Calcineurin and the Heartbeat, an evolving story. " and Buchholz etc., 2007, Cell Cycle.6 (1):16-9.“An Emerging role for Ca2+/calcineurin/NFAT signaling in cancerogenesis. ", whole It is hereby incorporated herein by.
As used herein, term " metabolic syndrome " for describe when occurring together, increase angiocardiopathy, apoplexy and The combination of the medical conditions of the risk of diabetes B.These illnesss include but not limited to central obesity (in body Portion and the additional weight on top)；Insulin resistance；Aging；It stress；Hormone change；Blood clotting is excessive；Dyslipidemia, bag It is relatively low to include HDL；Promote cardiopathic a type of LDL；And apolipoprotein (apolipoprotein) B100 rises.
The method for the treatment of and prevention
The present invention relates to calcium sensitivity enzyme CaMKII in primary HC by cAMP and glucagon and in vivo by pancreas height The discovery that blood glucose element and fasting are activated in a manner of calcium and IP3R dependences.The heredity of CaMKII lacks or inhibits to pass through influence The phosphorylation of FoxO1 blocks its core transposition, damages the decomposition of glycogen and gluconeogenesis of fasting and glucagon/cAMP inductions, And blood glucose level is reduced, and constitutive activity CaMKII has adverse effect.The containment that CaMKII lacks to glucose metabolism is made It is abolished with being transduceed by using composing type core FoxO1, so as to which the effect for indicating CaMKII shortages needs the core of FoxO1 Outer row.This identical approach also assists in the excessive HGP in obesity environment.These results disclose FoxO1 nuclear locations and liver grape The signal transduction path that calcium involved in sugared homeostasis mediates.
The present invention also relates to two kinds of drug targets of instruction-referred to as liver calcium transport bodies of IP3 receptors and referred to as calcium tune nerve phosphorus The liver phosphatase of sour enzyme-the discovery that can be of great value in this microhabitat of inhibitor, be included in the model of obesity In verification.It has recently found that the new role of IP3 receptors and calcineurin in terms of hepatic glucose generates (HGP), described Hepatic glucose is generated in obesity and diabetes B by overactivity.When in fasting or obesity, glucagon is lived Change release of the calcium of IP3 receptors and IP3R inductions from endoplasmic reticulum to cytosol.The calcium of release then activates 2 kinds of calcium sensitivity enzymes. One of described enzyme is CaMKII.The activation of CaMKII is required, because it promotes FoxO1 to enter in core, this is then induced The gene of HGP and partly cause the hyperglycemia in mouse obesity models and metabolic disorder.Calcineurin is Another enzyme activated by the calcium discharged.Calcineurin is a kind of another transcription factor dephosphorylation for making referred to as Crtc2, Thus it is promoted into the phosphatase in core.Crtc2 works together with FoxO1 with the gene of HGP in induced obesity.Cause This, as inhibiting CaMKII, inhibits the hyperglycemia in Crtc2 containment mouse obesity models.Therefore, the present invention carries Exploitation for IP3 receptors and Calcineurin inhibitors and in preclinical obesity and insulin resistance model to it Test.
Finally, the present invention relates to the discoveries that p38 and MK2/3 inhibitor can also be of great value in this microhabitat.
The present invention provide the method for the metabolic disorder of subject that treats or prevents, treat or prevent with metabolic disorder by The method that the method for the coronary artery disease of examination person and the hepatic glucose of reduction subject generate.The present invention is provided to subject Metabolic disorder treatment and/or prevention, subject to suffering from metabolic disorder coronary artery disease treatment and/or prevention And the method that the hepatic glucose of subject generates is reduced, the treatment and/or prevention and method are by described tested Person's application inhibit or reduce CaMKII, IP3R (include but not limited to IP3R1, IP3R2 and IP3R3), calcineurin, P38, MK2/3 or any combination thereof the compound of activity carry out.
The present invention also provides the compound of the activity of identification inhibition CaMKII or the activity and/or activation of reduction CaMKII Method.Therefore, the present invention is also provided to preventing the CaMKII of the metabolic disorder of obesity, type 1 diabetes and diabetes B Inhibitor screening, exploitation and test.The present invention further provides to combine to prevent with other inhibitor obesity, Screening, exploitation and the test of the inhibitor of the CaMKII of the metabolic disorder of type 1 diabetes and diabetes B, other inhibition Agent as but be not limited to IP3R (include but not limited to IP3R1, IP3R2 and IP3R3), calcineurin, p38 and/or MK2/3 Inhibitor.
The present invention is also provided to (being wrapped to prevent the IP3R of the metabolic disorder of obesity, type 1 diabetes and diabetes B Include but be not limited to IP3R1, IP3R2 and IP3R3) inhibitor and calcineurin inhibitor screening, exploitation and survey Examination.The present invention further provides to combining to prevent obesity, type 1 diabetes and diabetes B with other inhibitor The suppression of the inhibitor and/or calcineurin of the IP3R (including but not limited to IP3R1, IP3R2 and IP3R3) of metabolic disorder Screening, exploitation and the test of preparation, other inhibitor as but be not limited to CaMKII, calcineurin, p38 and/or MK2/3 inhibitor.
The present invention also provide to prevent the p38 of the metabolic disorder of obesity, type 1 diabetes and diabetes B and Screening, exploitation and the test of the inhibitor of MK2/3.The present invention further provides to combining to prevent fertilizer with other inhibitor Screening, exploitation and the test of the inhibitor of the p38 and MK2/3 of the metabolic disorder of fat disease, type 1 diabetes and diabetes B, it is described Other inhibitor as but be not limited to CaMKII, IP3R (include but not limited to IP3R1, IP3R2 and IP3R3) and/or calcium tune nerve Inhibitors of phosphatases.
In some aspects, invention as described herein is based on following find：The inhibitor of CaMKII can treat obesity and lure The insulin resistance and hepatic glucose and fat metabolic disturbance of hair.
In some aspects, invention as described herein provides CaMKII inhibitor to improve fertilizer for the purpose of drug development Metabolic disorder and their consequence in fat disease, metabolic syndrome, type 1 diabetes and diabetes B.
In some aspects, invention as described herein provides to treat and diagnose as obesity, Metabolic syndrome are sought peace 2 types The metabolic disorder of the insulin resistant state of diabetes, including cardiopathic method.
In some aspects, the present invention relates to the hepatic glucoses mediated with liver CaMKII in glucagon to generate in (HGP) The relevant result of study of effect.
As described herein, CaMKII activated in primary hepatocyte by glucagon and in vivo by glucagon and Fasting activates.The heredity of liver CaMKII lacks or inhibits to reduce blood glucose level, contains HGP gene G6pc and Pck1, reduces glycogen It exhausts, and blocks the core transposition of HGP transcription factors FoxO1.On the contrary, constitutive activity CaMKII induces G6pc and Pck1, stimulate Glucose generates, and raises blood glucose level.The containment that CaMKII lacks to glucose metabolism is acted on is given up by composing type core FoxO1 It removes, so as to indicate that the effect that CaMKII lacks can need to arrange FoxO1 outside core.
A kind of molecular pathways that by CaMKII are regulated and controled of the result identification as described herein in terms of HGP is controlled.
In some aspects, the present invention is related to finding fasting and glucagon activation liver CaMKII.In some aspects, originally Invention is to finding that CaMKII stimulates hepatic glucose generation related.In some aspects, the present invention is with having found that CaMKII promotes FoxO1's Nuclear location is related to activation.In some aspects, the glucose when present invention is with having found that CaMKII lacks, which generates, undermined needs core Outer row FoxO1 is related.In some aspects, method described herein is suitable for treatment and diagnosis of heart failure, because overactivity CaMKII has involved in heart failure.
The disclosure provides the method for treating and/or preventing metabolic disorder.On the one hand, the disclosure provide it is a kind for the treatment of or Prevent the method for the metabolic disorder of subject in need, the described method includes apply therapeutically effective amount to the subject The inhibitor of CaMKII, so as to treat or prevent the illness.On the other hand, it is in need to provide a kind for the treatment of or prevention for the disclosure Subject metabolic disorder method, the described method includes to the subject apply therapeutically effective amount IP3R1, IP3R2 And/or the inhibitor of IP3R3, so as to treat or prevent the illness.On the other hand, the disclosure provides a kind for the treatment of or prevention and has The method of the metabolic disorder of the subject needed, the described method includes the calcium tune nerves that therapeutically effective amount is applied to the subject The inhibitor of phosphatase, so as to treat or prevent the illness.On the other hand, it is in need to provide a kind for the treatment of or prevention for the disclosure Subject metabolic disorder method, the described method includes to the subject apply therapeutically effective amount p38 inhibitor, So as to treat or prevent the illness.On the other hand, the disclosure provides a kind of metabolism for treating or preventing subject in need The method of illness, the described method includes the inhibitor for the MK2/3 that therapeutically effective amount is applied to the subject, so as to treat or in advance Prevent the illness.On the other hand, the disclosure provides a kind of method for the metabolic disorder for treating or preventing subject in need, institute Stating method includes applying CaMKII, IP3R1, IP3R2, IP3R3, the calcium tune nerve phosphoric acid of therapeutically effective amount to the subject Enzyme, p38, MK2/3 or any combination thereof inhibitor, so as to treat or prevent the illness.
On the one hand, the disclosure provides a kind of method for the metabolic disorder for treating or preventing subject, and the described method includes drops The activity of low CaMKII, so as to treat or prevent the illness.On the one hand, the disclosure provides a kind for the treatment of or prevention subject's The method of metabolic disorder, wherein the illness is induced by obesity, the described method includes the activity for reducing CaMKII, so as to treat Or the prevention illness.On the one hand, the disclosure provides a kind of method for the metabolic disorder for treating or preventing subject, wherein described Illness is not induced by obesity, the described method includes the activity for reducing CaMKII, so as to treat or prevent the illness.
In one embodiment, illness is selected from by type 1 diabetes, diabetes B, insulin resistance and metabolic syndrome The group of composition.
In one embodiment, treating or preventing influences the decomposition of glycogen or gluconeogenesis of subject.In another embodiment party In case, the decomposition of glycogen or gluconeogenesis for not influencing subject are treated or prevented.
In one embodiment, the hepatic glucose generation for reducing subject, hyperglycaemia, fatty liver, pancreas are treated or prevented Island element resistance, the relevant inflammation of insulin resistance, the relevant dyslipidemia of insulin resistance or any combination thereof.In another implementation In scheme, the hepatic glucose generation for increasing subject, hyperglycaemia, fatty liver, insulin resistance, insulin resistance are treated or prevented The relevant dyslipidemia of relevant inflammation, insulin resistance or any combination thereof.In another embodiment, treatment or prevention pair The hepatic glucose generation of subject, hyperglycaemia, fatty liver, insulin resistance, the relevant inflammation of insulin resistance, insulin resistance Relevant dyslipidemia or any combination thereof without influence.
In one embodiment, treating or preventing reduces phosphorylation and/or the activation of CaMKII.In another embodiment In, treat or prevent the phosphorylation for inhibiting CaMKII.In another embodiment, the activity for inhibiting CaMKII is treated or prevented And/or activation.In one embodiment, treating or preventing increases phosphorylation and/or the activation of CaMKII.In another embodiment party In case, the activity for increasing CaMKII and/or activation are treated or prevented.
In one embodiment, the activity of CaMKII is the activity of glucagon induction.
In one embodiment, treating or preventing reduces or inhibits G6pc and/or Pck1 in the cell of subject Expression.In one embodiment, the expression for increasing G6pc and/or Pck1 in the cell of subject is treated or prevented.
In another embodiment, the FoxO1 protein contents in the core for reducing the cell of subject are treated or prevented. In another embodiment, the FoxO1 protein contents in the core for increasing the cell of subject are treated or prevented.
In another embodiment, treat or prevent reduce or inhibit FoxO1 protein amino acid S284, S295, S326, S467, S475, S246, S253, S413 or S415 or any combination thereof phosphorylation.In another embodiment, treat Or prevention increase FoxO1 protein amino acid S284, S295, S326, S467, S475, S246, S253, S413 or S415 or The phosphorylation of any combination thereof.
On the other hand, the disclosure provides a kind of method for the coronary artery disease for treating or preventing subject, the method Activity including the CaMKII for reducing the subject, so as to treat or prevent the coronary artery disease.The disclosure provides one The method that kind treats or prevents the coronary artery disease of the subject with metabolic disorder, it is described tested the described method includes increasing The activity of the CaMKII of person, so as to treat or prevent the coronary artery disease.
In one embodiment, illness is selected from by type 1 diabetes, diabetes B, insulin resistance and metabolic syndrome The group of composition.
In another embodiment, treating or preventing influences the decomposition of glycogen or gluconeogenesis of subject.In another embodiment party In case, the decomposition of glycogen or gluconeogenesis for not influencing subject are treated or prevented.
In one embodiment, the hepatic glucose generation for reducing subject, hyperglycaemia, fatty liver, pancreas are treated or prevented Island element resistance, the relevant inflammation of insulin resistance, the relevant dyslipidemia of insulin resistance or any combination thereof.In another implementation In scheme, the hepatic glucose generation for increasing subject, hyperglycaemia, fatty liver, insulin resistance, insulin resistance are treated or prevented The relevant dyslipidemia of relevant inflammation, insulin resistance or any combination thereof.
In one embodiment, treating or preventing reduces phosphorylation and/or the activation of CaMKII.In another embodiment In, treat or prevent the phosphorylation for increasing CaMKII and/or activation.
In one embodiment, the activity of CaMKII is the activity of glucagon induction.In another embodiment, The activity of CaMKII is not the activity of glucagon induction.
In one embodiment, the expression for reducing G6pc and/or Pck1 in the cell of subject is treated or prevented. In another embodiment, the expression for increasing G6pc and/or Pck1 in the cell of subject is treated or prevented.
In one embodiment, the FoxO1 protein contents in the core for reducing the cell of subject are treated or prevented. In another embodiment, the FoxO1 protein contents in the core for increasing the cell of subject are treated or prevented.
In another embodiment, treat or prevent reduce amino acid S284, S295 of FoxO1 protein, S326, S467, S475, S246, S253, S413 or S415 or any combination thereof phosphorylation.In another embodiment, treatment or pre- It is anti-increase FoxO1 protein amino acid S284, S295, S326, S467, S475, S246, S253, S413 or S415 or its The phosphorylation of what combination.
In one embodiment, the CaMKII activity for including reducing in the macrophage of subject is treated or prevented. In another embodiment, the CaMKII activity included increasing in the macrophage of subject is treated or prevented.
In one embodiment, coronary artery disease is formed to atheroma and/or atherosclerosis is related. In another embodiment, method further comprises treating or preventing heart failure, hypertension and/or nephrosis.In another embodiment party In case, illness is related to advanced lesions macrophage apoptosis.In another embodiment, illness is related to patch necrosis.
In one embodiment, treating or preventing the coronary artery disease of the subject with metabolic disorder causes to mitigate Hyperinsulinemia and/or dyslipidemia.In another embodiment, the hat of the subject with metabolic disorder is treated or prevented Coronary disease causes to increase hyperinsulinemia and/or dyslipidemia.
In one embodiment, treating or preventing the coronary artery disease of the subject with metabolic disorder causes to mitigate Atheroma is formed and/or atherosclerosis.In another embodiment, treat or prevent tested with metabolic disorder The coronary artery disease of person causes to increase atheroma formation and/or atherosclerosis.
In one embodiment, the step for the inhibitor for including applying CaMKII protein to subject is treated or prevented Suddenly.In one embodiment, the table for the gene for including reducing coding CaMKII protein to subject's application is treated or prevented The step of antisense RNA or siRNA for reaching.In another embodiment, treating or preventing includes tying to subject's application specificity The step of closing the peptide or polypeptide of CaMKII protein.In another embodiment, treating or preventing is included to subject using small The step of molecule.In one embodiment, small molecule is the CaMKII inhibitor selected from the group being made up of：KN-93, smoke Lavendustin C, CK59, Ant-CaMKIINtide, KN62, DY9760e, Nocardiopsis K-252a, H89 dihydrochloride, PP1 Analog II1NM-PP1, eEF-2 kinase inhibitor NH125 and STO-609.
On the other hand, the disclosure provides a kind of method that hepatic glucose for reducing subject generates, and the described method includes drops The activity of low CaMKII, so as to cause the reduction of the hepatic glucose of the subject.On the other hand, the disclosure provides a kind of The method that the hepatic glucose of subject generates is reduced, the described method includes the activity for increasing CaMKII, so as to cause described tested The reduction of the hepatic glucose of person.
On the other hand, the disclosure provides a kind of method that hepatic glucose for increasing subject generates, and the described method includes increasings Add the activity of CaMKII, so as to cause the increase of the hepatic glucose of the subject.On the other hand, the disclosure provides a kind of Increase the method that the hepatic glucose of subject generates, the described method includes the activity for reducing CaMKII, so as to cause described tested The increase of the hepatic glucose of person.
In one embodiment, reduce hepatic glucose and generate the phosphorylation for reducing CaMKII and/or activation.In another reality It applies in scheme, reduces hepatic glucose and generate the phosphorylation for increasing CaMKII and/or activation.
In one embodiment, increase hepatic glucose and generate the phosphorylation for reducing CaMKII and/or activation.In another reality It applies in scheme, increases hepatic glucose and generate the phosphorylation for increasing CaMKII and/or activation.
In one embodiment, the activity of CaMKII is the activity of glucagon induction.
In one embodiment, reduce hepatic glucose and generate reduction G6pc and/or Pck1 in the cell of subject Expression.In another embodiment, reduce hepatic glucose and generate the table for increasing G6pc and/or Pck1 in the cell of subject It reaches.In one embodiment, increase hepatic glucose and generate the expression for reducing G6pc and/or Pck1 in the cell of subject. In another embodiment, increase hepatic glucose and generate the expression for increasing G6pc and/or Pck1 in the cell of subject.
In one embodiment, the hyperglycaemia, fatty liver, insulin for reducing hepatic glucose generation reduction subject resist Property, the relevant inflammation of insulin resistance, the relevant dyslipidemia of insulin resistance.In another embodiment In, reduce hepatic glucose generate increase the hyperglycaemia of subject, fatty liver, insulin resistance, the relevant inflammation of insulin resistance, The relevant dyslipidemia of insulin resistance or any combination thereof.In one embodiment, increase hepatic glucose generate reduce by The relevant inflammation of the hyperglycaemia of examination person, fatty liver, insulin resistance, insulin resistance, the relevant dyslipidemia of insulin resistance Or any combination thereof.In another embodiment, increase hepatic glucose and generate the hyperglycaemia for increasing subject, fatty liver, pancreas islet The relevant inflammation of plain resistance, insulin resistance, the relevant dyslipidemia of insulin resistance or any combination thereof.
In another embodiment, the FoxO1 protein in the core for the cell that hepatic glucose generates reduction subject is reduced Content.In another embodiment, reduce hepatic glucose generate reduce amino acid S284, S295 of FoxO1 protein, S326, S467, S475, S246, S253, S413 or S415 or any combination thereof phosphorylation.In another embodiment, liver Portugal is reduced Grape sugar generates the FoxO1 protein contents in the core for increasing the cell of subject.In another embodiment, hepatic glucose is reduced Generate amino acid S284, S295, S326, S467, S475, S246, S253, S413 or the S415 or its for increasing FoxO1 protein Any combination of phosphorylation.
In another embodiment, the FoxO1 protein in the core for the cell that hepatic glucose generates reduction subject is increased Content.In another embodiment, increase hepatic glucose generate reduce amino acid S284, S295 of FoxO1 protein, S326, S467, S475, S246, S253, S413 or S415 or any combination thereof phosphorylation.In another embodiment, liver Portugal is increased Grape sugar generates the FoxO1 protein contents in the core for increasing the cell of subject.In another embodiment, hepatic glucose is increased Generate amino acid S284, S295, S326, S467, S475, S246, S253, S413 or the S415 or its for increasing FoxO1 protein Any combination of phosphorylation.
In one embodiment, reducing hepatic glucose and generating includes reducing coding CaMKII protein to subject's application Gene expression antisense RNA the step of.In one embodiment, increase hepatic glucose to generate including applying to subject The step of reducing the antisense RNA of the expression of the gene of coding CaMKII protein.
In another embodiment, reducing hepatic glucose and generating is included to subject's application specific binding CaMKII albumen The step of peptide or polypeptide of matter.In another embodiment, increase hepatic glucose to generate including tying to subject's application specificity The step of closing the peptide or polypeptide of CaMKII protein.
In another embodiment, the step of hepatic glucose is generated including applying small molecule to subject is reduced.Another In embodiment, increase the step of hepatic glucose is generated including applying small molecule to subject.
In one embodiment, small molecule is the CaMKII inhibitor selected from the group being made up of：KN-93, grass is smoked Rhzomorph C, CK59, Ant-CaMKIINtide, KN62, DY9760e, Nocardiopsis K-252a, H89 dihydrochloride, PP1 classes Like object II1NM-PP1, eEF-2 kinase inhibitor NH125 and STO-609.
On the one hand, the disclosure provides a kind of method for the metabolic disorder for treating or preventing subject, and the described method includes drops The activity of low IP3R1, IP3R2 or IP3R3, so as to treat or prevent the illness.The disclosure provide it is a kind of treat or prevent by The method of the metabolic disorder of examination person, wherein the illness is induced by obesity, the described method includes reduce IP3R1, IP3R2 or The activity of IP3R3, so as to treat or prevent the illness.The disclosure provides a kind of metabolic disorder for treating or preventing subject Method, wherein the illness not induced by obesity, the described method includes reduce IP3R1, IP3R2 or IP3R3 activity, from And treat or prevent the illness.
On the other hand, the disclosure provides a kind of method for the metabolic disorder for treating or preventing subject, the described method includes The activity of calcineurin is reduced, so as to treat or prevent the illness.On the other hand, the disclosure provide it is a kind for the treatment of or Prevent the method for the metabolic disorder of subject, wherein the illness is induced by obesity, the described method includes reduce calcium tune nerve The activity of phosphatase, so as to treat or prevent the illness.On the other hand, the disclosure provides a kind for the treatment of or prevention subject's The method of metabolic disorder, wherein the illness is not induced by obesity, the described method includes reduce calcineurin Activity, so as to treat or prevent the illness.
In one embodiment, illness was selected from by type 1 diabetes, diabetes B, insulin resistance, obesity and generation Thank to the group of syndrome composition.
In one embodiment, treating or preventing influences the decomposition of glycogen or gluconeogenesis of subject.In another embodiment party In case, the decomposition of glycogen or gluconeogenesis for not influencing subject are treated or prevented.
In one embodiment, the hepatic glucose generation for reducing subject, hyperglycaemia, fatty liver, pancreas are treated or prevented Island element resistance, the relevant inflammation of insulin resistance, the relevant dyslipidemia of insulin resistance or any combination thereof.In another implementation In scheme, the hepatic glucose generation for increasing subject, hyperglycaemia, fatty liver, insulin resistance, insulin resistance are treated or prevented The relevant dyslipidemia of relevant inflammation, insulin resistance or any combination thereof.
In one embodiment, treat or prevent include to subject apply IP3R1 protein, IP3R2 protein, IP3R3 protein or any combination thereof inhibitor the step of.In one embodiment, inhibitor is smooth Spongistatin C. In another embodiment, inhibitor is 2-APB.In another embodiment, inhibitor is caffeine (caffeine).
In one embodiment, treating or preventing includes reducing coding IP3R1 protein, IP3R2 to subject's application Protein or IP3R3 protein or any combination thereof gene expression antisense RNA or siRNA the step of.In another implementation In scheme, treating or preventing is included to subject application specific binding IP3R1 protein, IP3R2 protein or IP3R3 albumen Matter or any combination thereof peptide or polypeptide the step of.
In one embodiment, the step for the treatment of or preventing including applying small molecule to subject.In an embodiment party In case, small molecule is IP3R1 protein inhibitors, IP3R2 protein inhibitors or IP3R3 protein inhibitors.In a reality It applies in scheme, small molecule is smooth Spongistatin C.In another embodiment, small molecule is 2-APB.In another embodiment, Small molecule is caffeine.
In one embodiment, the step for the inhibitor for including applying calcineurin to subject is treated or prevented Suddenly.In one embodiment, the inhibitor of calcineurin is cyclosporin A (cyclosporin A).In another reality It applies in scheme, the inhibitor of calcineurin is Elidel (pimecrolimus).In another embodiment, calcium tune The inhibitor of neural phosphatase is tacrolimus (tacrolimus).
In one embodiment, treating or preventing includes reducing coded Ca tune nerve phosphatase protein to subject's application The step of antisense RNA or siRNA of the expression of the gene of matter.In another embodiment, treating or preventing is included to subject The step of using the peptide or oligopeptides or polypeptide for specifically binding calcineurin protein.In one embodiment, it is few Peptide is cyclosporin A.
In one embodiment, the step for the treatment of or preventing including applying small molecule to subject.In another embodiment party In case, small molecule is Calcineurin inhibitors.In one embodiment, small molecule is Elidel.In another reality It applies in scheme, small molecule is tacrolimus.
On the other hand, the disclosure provides a kind of method for the metabolic disorder for treating or preventing subject, the described method includes The activity of p38 is reduced, so as to treat or prevent the illness.On the other hand, the disclosure provides a kind for the treatment of or prevention subject Metabolic disorder method, wherein the illness is induced by obesity, the described method includes the activity for reducing p38, so as to treat Or the prevention illness.On the other hand, the disclosure provides a kind of method for the metabolic disorder for treating or preventing subject, wherein institute It states illness not to be induced by obesity, the described method includes the activity for reducing p38, so as to treat or prevent the illness.
On the other hand, the disclosure provides a kind of method for the metabolic disorder for treating or preventing subject, the described method includes The activity of MK2/3 is reduced, so as to treat or prevent the illness.On the other hand, it is tested to provide a kind for the treatment of or prevention for the disclosure The method of the metabolic disorder of person, wherein the illness is induced by obesity, the described method includes the activity for reducing MK2/3, thus Treat or prevent the illness.On the other hand, the disclosure provides a kind of method for the metabolic disorder for treating or preventing subject, Described in illness not induced by obesity, the described method includes reduce MK2/3 activity, so as to treat or prevent the disease Disease.
In one embodiment, illness was selected from by type 1 diabetes, diabetes B, insulin resistance, obesity and generation Thank to the group of syndrome composition.
In one embodiment, treating or preventing influences the decomposition of glycogen or gluconeogenesis of subject.In another embodiment party In case, the decomposition of glycogen or gluconeogenesis for not influencing subject are treated or prevented.
In one embodiment, the hepatic glucose generation for reducing subject, hyperglycaemia, fatty liver, pancreas are treated or prevented Island element resistance, the relevant inflammation of insulin resistance, the relevant dyslipidemia of insulin resistance or any combination thereof.In another implementation In scheme, the hepatic glucose generation for increasing subject, hyperglycaemia, fatty liver, insulin resistance, insulin resistance are treated or prevented The relevant dyslipidemia of relevant inflammation, insulin resistance or any combination thereof.
In one embodiment, the step for the treatment of or preventing the inhibitor including applying p38 to subject.In a reality It applies in scheme, the inhibitor of p38 is SB203580.In another embodiment, the inhibitor of p38 is SB202190.Another In embodiment, the inhibitor of p38 is SB239063.
In one embodiment, treating or preventing includes the gene to subject's application reduction coding p38 protein The step of antisense RNA or siRNA of expression.In another embodiment, treating or preventing is included to subject's application specificity With reference to p38 protein peptide or polypeptide the step of.In another embodiment, treating or preventing includes applying small point to subject The step of son.In one embodiment, small molecule is p38 inhibitor.In one embodiment, small molecule is SB203580.In another embodiment, small molecule is SB202190.In another embodiment, small molecule is SB239063.
In one embodiment, the step for the treatment of or preventing the inhibitor including applying MK2/3 to subject.At one In embodiment, the inhibitor of MK2/3 is Hsp25 kinase inhibitors.
In one embodiment, the gene for including that coding MK2/3 protein is reduced to subject's application is treated or prevented Expression antisense RNA or siRNA the step of.In another embodiment, treating or preventing is included to subject's application specifically The step of peptide or polypeptide of property combination MK2/3 protein.In one embodiment, peptide is Hsp25 kinase inhibitors.At one In embodiment, the step for the treatment of or prevention including applying small molecule to subject.In one embodiment, small molecule is MK2/3 inhibitor.
Screening technique
The disclosure provides to identify the combination of the compound or compound of the metabolic disorder for treating or preventing subject Method.The disclosure also provides to identify the compound for the coronary artery disease for treating or preventing the subject with metabolic disorder Or the method for the combination of compound.In one embodiment, illness is induced by obesity.In another embodiment, illness Not induced by obesity.The disclosure also provides to identify the compound or compound that the hepatic glucose for reducing subject generates Combination method.
The disclosure, which also provides to identify, inhibits or reduces the activity of CaMKII and/or the compound of activation or compound The method of combination.The disclosure, which also provides to identify, inhibits or reduces IP3R (including but not limited to IP3R1, IP3R2 and IP3R3) Activity and/or activation compound or compound combination method.The disclosure, which also provides to identify, inhibits or reduces calcium The method for adjusting the combination of the neural activity of phosphatase and/or the compound of activation or compound.The disclosure also provides to identify Inhibit or reduce the method for the combination of the activity of p38 and/or the compound or compound of activation.The disclosure also provides to identify Inhibit or reduce the method for the combination of the activity of MK2/3 and/or the compound or compound of activation.
The disclosure also provide to identify inhibit CaMKII, IP3R (include but not limited to IP3R1, IP3R2 and IP3R3), Calcineurin, p38, MK2/3 or any combination thereof activity and/or activation compound or compound combination side Method.The disclosure, which also provides to identify, reduces CaMKII, IP3R (including but not limited to IP3R1, IP3R2 and IP3R3), calcium tune god Through phosphatase, p38, MK2/3 or any combination thereof activity compound or compound combination method.
The disclosure provides a kind of method of the compound for the activity for inhibiting CaMKII for identification, and the described method includes a) make Cell is contacted with CaMKII fusion proteins, wherein the CaMKII fusion proteins are included in the acceptor fluorescence group albumen of an end Matter and the donor fluorophore protein in another end；And b) the absence and presence of test compound in the case of measure FRET efficiency, wherein compared to the FRET efficiency in the case of there is no the test compound, in the test compound In the presence of the FRET efficiency larger instruction test compound inhibit CaMKII activity (referring to Takao etc., 2005；With Kwok etc., 2008, be incorporated herein in its entirety by reference).
The disclosure provides a kind of method of the compound for the activity for inhibiting CaMKII for identification, and the described method includes a) make Cell is contacted with CaMKII fusion proteins, wherein the CaMKII fusion proteins are included in the acceptor fluorescence group albumen of an end Matter and the donor fluorophore protein in another end；And b) the absence and presence of test compound in the case of measure institute Donor protein matter and the ratio of the receptor protein are stated, wherein compared in the case of there is no the test compound Ratio, the ratio in the presence of the test compound reduce the activity that the instruction test compound inhibits CaMKII (referring to Takao etc., 2005；With Kwok etc., 2008, be incorporated herein in its entirety by reference).
In one embodiment, acceptor fluorescence group protein is selected from the group being made up of：mOrange、 MStrawberry, Venus, yellow fluorescence protein, cyan fluorescent protein, red fluorescent protein and green fluorescent protein.Another In embodiment, donor protein matter is selected from the group being made up of：MOrange, mStrawberry, Venus, yellow fluorescence egg In vain, cyan fluorescent protein, red fluorescent protein and green fluorescent protein (referring to Takao etc., 2005；With Kwok etc., 2008, It is incorporated herein in its entirety by reference).
In one embodiment, cell be HEK293T cells, liver cell, U2OS cells, HeLa cells, mouse embryo into Fibrocyte (MEF) or macrophage.In another embodiment, cell from Insr-/- mouse, Camk2g-/- mouse, Foxo1-/- mouse, db/db mouse, ob/ob mouse, p38-/- mouse, non-obese diabetes (NOD) mouse, feeding are high in fat The mouse of meals or the mouse of streptozotocin processing.In another embodiment, cell is from expression mutation FoxO1 protein Mouse.In one embodiment, be mutated FoxO1 protein be included in S284, S295, S326, S467, S475, S246, Alanine substitution at S253, S413 or S415 or in S284, S295, S326, S467, S475, S246, S253, S413 or At S415 aspartic acid substitution or any combination thereof.
In one embodiment, cell is made to be subjected to ER stress.In another embodiment, cell glucagon, 8- bromine cAMP, H89 dihydrochloride, smooth Spongistatin C, forskolin, saturated fatty acid or any combination thereof processing.
On the other hand, the disclosure provides a kind of compound for the activity for inhibiting IP3R1, IP3R2 or IP3R3 for identification Method is contacted the described method includes cell a) is made with test compound；And IP3R1, IP3R2 or IP3R3 activity b) are measured, In activity compared to IP3R1, IP3R2 or IP3R3 in the case of there is no the compound, there are the compounds In the case of the activity of IP3R1, IP3R2 or IP3R3 reduce and indicate that the compound is IP3R1, IP3R2 or IP3R3 respectively Inhibitor.
In one embodiment, by after being stimulated with the derivant of IP3, discharging to the calcium in the cytosol of cell To measure activity.In one embodiment, discharged by the increase of the fluorescence of kytoplasm liquid calcium dyestuff to measure calcium.In a reality It applies in scheme, kytoplasm liquid calcium dyestuff is Fluo-3.
On the other hand, the disclosure provides a kind of side of the compound for the activity for inhibiting calcineurin for identification Method is contacted the described method includes cell a) is made with test compound；And Calcineurin Activities b) are measured, wherein comparing In the calcineurin in the case of there is no the compound activity in the case of there are the compound The activity of calcineurin reduces the inhibitor for indicating that the compound is calcineurin.
In one embodiment, phosphatase activity is detected by using calcineurin peptide substrate to measure calcium tune The activity of neural phosphatase.In one embodiment, cell is HEK293T cells, liver cell, U2OS cells, HeLa cells Or rat embryo fibroblast cell (MEF).In another embodiment, cell is from db/db mouse, ob/ob mouse, non-obese sugar The mouse of sick (NOD) mouse of urine, the mouse of feeding high fat diet or streptozotocin processing.
In one embodiment, cell glucagon, H89 dihydrochlorides, insulin, forskolin, ER stress Derivant, tunicamycin, saturated fatty acid or any combination thereof processing.
On the other hand, the disclosure provides a kind of method of the compound for the activity for inhibiting p38 for identification, the method bag Including a) makes cell be contacted with test compound；And p38 kinase activities b) are measured, wherein compared to there is no the compounds In the case of p38 kinase activity, the kinase activity of the p38 in the case of there are the compound reduces instruction describedization Close the inhibitor that object is p38.On the other hand, the disclosure provides a kind of side of the compound for the activity for inhibiting MK2/3 for identification Method is contacted the described method includes cell a) is made with test compound；And b) measure MK2/3 kinase activities, wherein compared to The kinase activity of MK2/3 in the case of there is no the compound, MK2/3's in the case of there are the compound swashs Enzymatic activity reduces the inhibitor for indicating that the compound is MK2/3.
In one embodiment, p38 kinase activities are measured using p38 specific peptides.In one embodiment, use MK2/3 specific peptides measure MK2/3 kinase activities.
In one embodiment, cell is HEK293T cells, liver cell, U2OS cells, HeLa cells, macrophage Or rat embryo fibroblast cell (MEF).In another embodiment, cell is from Insr-/- mouse, db/db mouse, ob/ob The mouse that mouse, non-obese diabetes (NOD) mouse, the mouse of feeding high fat diet or streptozotocin are handled.In another reality It applies in scheme, cell glucagon, 8- bromine cAMP, H89 dihydrochloride, forskolin, saturated fatty acid or any combination thereof Processing.
On the one hand, method is included to the animal as metabolic disorder or the model of angiocardiopathy (such as coronary artery disease) Using test compound or test the combination of compound and determine compared to the animal not being processed as, the compound or Whether the combination of compound improves metabolic function and/or cardiovascular function.
The present invention is provided to identify to can be used for the metabolic disorder for treating or preventing subject, treatment or prevention with metabolism The method for the compound that the coronary artery disease of the subject of illness and/or the hepatic glucose of reduction subject generate.The present invention The method for providing to identify the activity for inhibiting CaMKII or reduce the phosphorylation of CaMKII and/or the compound of activation.This hair It is bright provide to identify the activity for inhibiting IP3R (include but not limited to IP3R1, IP3R2 and IP3R3) or reduce IP3R (including but Be not limited to IP3R1, IP3R2 and IP3R3) phosphorylation and/or activation compound method.The present invention is provided to identify suppression The activity of calcineurin processed or the method for reducing the phosphorylation of calcineurin and/or the compound of activation.This Invention provides the method identified the activity for inhibiting p38 or reduce the phosphorylation of p38 and/or the compound of activation.The present invention The method for providing to identify the activity for inhibiting MK2/3 or reduce the phosphorylation of MK2/3 and/or the compound of activation.
Method may include characterization test compound or medicament, and (such as peptide (such as antibody or its segment), small molecule or nucleic acid are (such as SiRNA or antisense RNA) or other medicaments).
In one embodiment, compound can be peptide fragment.Segment is included between about 8 amino acid and about 100 ammonia Between base acid and all possible amino acid length including about 8 amino acid and about 100 amino acid, such as following length： Between about 10 amino acid and about 100 amino acid, between about 15 amino acid and about 100 amino acid, between Between about 20 amino acid and about 100 amino acid, between about 35 amino acid and about 100 amino acid, between about 40 Between a amino acid and about 100 amino acid, between about 50 amino acid and about 100 amino acid, between about 70 ammonia Between base acid and about 100 amino acid, between about 75 amino acid and about 100 amino acid or between about 80 amino acid Between about 100 amino acid.These peptide fragments are commercially available or synthesized by liquid phase or solid phase synthesis process (Atherton etc., (1989) Solid Phase Peptide Synthesis:a Practical Approach.IRL Press,Oxford,England).Peptide fragment can be separated from natural origin, carried out genetically engineered or chemically made It is standby.These methods are well known in the art.
Compound can be protein, such as antibody (monoclonal antibody, polyclonal antibody, humanized antibody, chimeric antibody or complete Human antibody) or its binding fragment.Antibody fragment can be the antibody formation in addition to overall length form and remove by engineered Outside antibody fragment, also include being present in the part in full length antibody or component.Antibody fragment may include but be not limited to scFv (scFv), double-chain antibody, Fv and (Fab ')2, three chain antibodies, the combination of Fc, Fab, CDR1, CDR2, CDR3, CDR, variable region, Four chain antibodies, difunctional hybrid antibody, framework region, constant region etc. are (referring to Maynard etc., (2000) Ann.Rev.Biomed.Eng.2:339-76；Hudson(1998)Curr.Opin.Biotechnol.9:395-402).Antibody It is commercially available, customization generate or according in this field establish method for target antigen come synthesize (Janeway etc., (2001) Immunobiology, the 5th edition, Garland Publishing).
Compound may be selected from comprising following group：siRNA；RNA interfering or RNAi；dsRNA；Rna plymerase iii transcription DNA；Ribozyme；With the antisensenucleic acids that can be RNA, DNA or artificial nucleic acid.Including antisense DNA, RNA and DNA/RNA molecules it is anti- Oligonucleotide plays a part of directly to block mRNA to translate by the mRNA with reference to targeting and prevention protein translation.It can be such as Pass through antisense oligonucleotides (Dallas etc., (2006) of the synthesis of conventional phosphoric acid diester technology at least about 15 bases Med.Sci.Monit.12(4):RA67-74；Kalota etc., (2006) Handb.Exp.Pharmacol.173:173-96； Lutzelburger etc., (2006) Handb.Exp.Pharmacol.173:243-59).Antisense base sequences include but unlimited In morpholinyl nucleotide sequence, 2 '-O- methyl polynucleotides, DNA, RNA etc..
SiRNA includes duplex structure, contains about 15 to about 50 base-pairs, for example, about 21 to about 25 base-pairs, and With nucleotide sequence identical with the target gene or RNA expressed in the cell or almost identical.SiRNA is included and irrigated by standard Just RNA chains and the complementary anti-sense RNA chains that gloomy-Crick (Watson-Crick) base pairing interaction is annealed together. Positive-sense strand includes the nucleotide sequence being substantially the same with the nucleotide sequence contained in target miRNA molecule.With containing in said target mrna Target sequence " being substantially the same " refers to that nucleotide sequence and target sequence have about 3% or smaller difference.The positive-sense strand and antisense of siRNA Chain, which may make up two complementary single strand RNA molecules or may make up two of which complementary portion, carries out base pairing and by single-stranded " hair The single molecule that folder " area is covalently attached.Referring also to McMnaus and Sharp (2002) Nat Rev Genetics, 3:737- 47 and Sen and Blau (2006) FASEB J., 20:1293-99, entire disclosure are hereby incorporated herein by.
SiRNA can be the RNA changed, the difference when adding, lack, substitute and/or changes one or more nucleotide In naturally occurring RNA.The change may include the internal nucleotide of one or more such as to the end of siRNA or to siRNA Addition non-nucleotide substance takes into modification resistant to nuclease digestion enforcement siRNA or with deoxyribonucleotide For one or more of siRNA nucleotide.One or two chain of siRNA can also include 3 ' prominent (overhang).Such as this Used in text, 3 ' the prominent at least one unpaired nucleotides for referring to extend from 3 ' ends of duplex RNA chain.For example, siRNA can Comprising at least one length be 1 to about 6 nucleotide (it includes ribonucleotide or deoxyribonucleotide) or length is 1 3 ' the protrusions that about 5 nucleotide or length be 1 to about 4 nucleotide or length is about 2 to about 4 nucleotide.For example, It is prominent that each chain of siRNA can include 3 ' with two thymidylic acids (" TT ") or two uridylic acids (" uu ").
SiRNA can be generated chemically or with biological mode or (such as can joined from recombinant plasmid or viral vectors expression See U.S. Patent number 7,294,504 and U.S. Patent number 7,422,896, entire disclosure is herein incorporated by reference this Text).Illustrative methods for generating and testing dsRNA or siRNA molecule are described in the U.S. Patent Application Publication of Gewirtz Number 2002/0173478, the United States Patent (USP) Shen of the U.S. Patent Application Publication No. 2007/0072204 of Hannon etc. and Reich etc. Please be in publication No. 2004/0018176, the entire disclosure of the announcement is hereby incorporated herein by.
The DNA of rna plymerase iii transcription contains promoter, such as U6 promoters.These DNA can be transcribed in cell The children purpura nephritis that may act as siRNA or linear rna is generated, the siRNA or linear rna may act as antisense RNA.Compound can Containing ribonucleotide, deoxyribonucleotide, synthesizing ribonucleotide or any suitable combination so that target RNA and/or gene are suppressed System.In addition, the nucleic acid of these forms can be single-stranded, double-strand, three chains or four chains.(see, for example, Bass (2001) Nature, 411, 428429；Elbashir etc., (2001) Nature, 411,494498；With PCT Publication WO00/44895, WO01/36646, WO99/32619、WO00/01846、WO01/29058、WO99/07409、WO00/44914)。
Compound can be conjugated protein and destroy its function or enhance the small molecule of its function on the contrary.Small molecule It is a different set of synthesis and the natural materials usually with low molecular weight.They can from natural origin (such as plant, fungi, Microorganism etc.) separation, it is commercially available and/or obtain or synthesized with library or aggregate form.It can be by combinatorial libraries Electron screening (in silico screening) or high-throughput (HTP) screen to identify candidate small molecule.Most conventional drug (such as aspirin (aspirin), penicillin and many chemotherapeutants) is small molecule, commercially available, chemically synthesize or Can (Werner etc., (2006) Brief Funct.Genomic Proteomic5 be obtained from random or combinatorial libraries as described below (1):32-6)。
In addition to agonist, understanding and the sequence to the primary sequence of target molecule and protein known to function Similitude can also provide the information of the inhibitor or antagonist on target protein.By using X-ray crystallography, in Sub- diffraction, NMR spectrum art and other technologies for structure determination determine the structure feature of protein, to agonist and The identification and screening of antagonist are further promoted.These technologies provide the rational design or mirror to agonist and antagonist It is fixed.
Compound can be tested from the large-scale library screening with synthesis or native compound (referring to Wang etc., (2007) Curr Med Chem,14(2):133-55；Mannhold(2006)Curr Top Med Chem,6(10):1031-47；With Hensen(2006)Curr Med Chem 13(4):361-76).Numerous means be currently used in random and controlled syntheses be based on it is sugared, The compound of peptide and nucleic acid.Synthesize library of compounds available commercially from Maybridge Chemical Co. (Trevillet, Cornwall, UK), AMRI (Albany, NY), ChemBridge (San Diego, CA) and MicroSource (Gaylordsville,CT).Rare chemistry library can be obtained from Aldrich (Milwaukee, Wis.).Alternatively, it is in bacterium, true Bacterium, plant and animal form of extract native compound library can from such as Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.) is obtained or can readily generate.In addition, natural and synthetically produced library and compound are easy In being modified (Blondelle etc., (1996) Tib Tech14 by conventional chemical, physics and biochemistry means:60).
The method for being used to prepare molecular library is that well known and many libraries are commercially available in the art.Mesh in the present invention Marking library includes peptide library, randomized oligonucleotides library, synthesis organic combinatorial libraries etc..Degeneracy peptide library can be easy to solution Form, using as bacterial flagellum peptide display libraries or as phage display library fixed form prepare.Peptide ligand may be selected from The combinatorial libraries of peptide containing at least one amino acid.The library of class peptide and non-peptide composite part can be synthesized.It can further synthesize Containing the naturally occurring counterpart compared to them, the library of the non-peptide composite part of enzymatic degradation is less subjected to.Example Such as, library may also comprise but be not limited to peptide in (peptide-on-plasmid) library, synthesized micromolecule library, aptamer on plasmid Library, based in vitro translated library, polysome library, synthesis peptide library, neurotransmitter library and chemistry library.
The example in chemical synthesis library be described in it is following in：Fodor etc., (1991) Science 251:767-773； Houghten etc., (1991) Nature354:84-86；Lam etc., (1991) Nature354:82-84；Medynski,(1994) BioTechnology12:709-710；Gallop etc., (1994) J.Medicinal Chemistry37 (9):1233-1251； Ohlmeyer etc., (1993) Proc.Natl.Acad.Sci.USA90:10922-10926；Erb etc., (1994) Proc.Natl.Acad.Sci.USA91:11422-11426；Houghten etc., (1992) Biotechniques 13:412； Jayawickreme etc., (1994) Proc.Natl.Acad.Sci.USA91:1614-1618；Salmon etc., (1993) Proc.Natl.Acad.Sci.USA90:11708-11712；PCT Publication WO93/20242, October 14 1993 date； With Brenner etc., (1992) Proc.Natl.Acad.Sci.USA89:5381-5383.The example description of phage display library In following：Scott etc., (1990) Science249:386-390；Devlin etc., (1990) Science, 249:404-406； Christian, etc. (1992) J.Mol.Biol.227:711-718；Lenstra,(1992)J.Immunol.Meth.152: 149-157；Kay etc., (1993) Gene128:59-65；With PCT Publication WO94/18318.It is wrapped based in vitro translated library Include but be not limited to be described in it is following in those：PCT Publication WO91/05058；With Mattheakis etc., (1994) Proc.Natl.Acad.Sci.USA91:9022-9026。
It can be realized by any kind of commonly known method to library screening.See, for example, the open sieve to peptide library Choosing below with reference to document：Parmley and Smith, (1989) Adv.Exp.Med.Biol.251:215-218；Scott and Smith,(1990)Science249:386-390；Fowlkes etc., (1992) BioTechniques13:422-427； Oldenburg etc., (1992) Proc.Natl.Acad.Sci.USA89:5393-5397；Yu etc., (1994) Cell76:933- 945；Staudt etc., (1988) Science241:577-580；Bock etc., (1992) Nature355:564-566；Tuerk etc., (1992)Proc.Natl.Acad.Sci.USA89:6988-6992；Ellington etc., (1992) Nature355:850-852； U.S. Patent number 5,096,815；5,223,409；With 5,198,346, Ladner etc. is all belonged to；Rebar etc., (1993) Science263:671-673；With PCT Publication WO94/18318.
Also it can generate and screen small molecule combinatorial library.The combinatorial libraries of small organic compound are one or more various Property main points in terms of closely related analog different from each other set and synthesized using multistep method by organic technology.Combination Library includes the small organic compound of tremendous amount.A type of combinatorial libraries are by means of generating chemical array Prepared by Parallel Synthesis Procedure.Chemical array can be can be by them at Cartesian coordinate (Cartesian coordinate) In space address identification compound set and can be arranged so that each compound have common molecular core and one or Multiple varistructure diversity elements.Compound parallel generation in independent reaction vessel in the chemical array, In identify and track each compound by the space address of each compound.Parallel projects mixture and Parallel Synthesis Procedure Example be provided below：The U.S. sequence numbers 08/177,497 and 13 days July nineteen ninety-five that on January 5th, 1994 submits are announced Its corresponding PCT Publication patent application W095/18972 and the U.S. Patent number 5,712,171 authorized on January 27th, 1998 And its corresponding PCT Publication patent application W096/22529, the patent are hereby incorporated herein by accordingly.
In one non-limiting embodiment, non-peptide library can be screened, such as benzodiazepine * Boom (benzodiazepine) texts Storehouse is (see, for example, Bunin etc., (1994) Proc.Natl.Acad.Sci.USA91:4708-4712).It can also be used class peptide literary Storehouse, such as by Simon, (1992) Proc.Natl.Acad.Sci.USA89:Person described in 9367-9371.Ostresh etc. (1994),Proc.Natl.Acad.Sci.USA91:Another example in library, wherein peptide workable for 11138-11142 descriptions In amide functional base generate the combinatorial libraries of chemical conversion by permethylated.
Microcomputer modelling and search technique allow authenticating compound or improve the compound identified, the compound can be controlled Treat or prevention subject metabolic disorder, treat or prevent with metabolic disorder subject coronary artery disease, reduce by The hepatic glucose of examination person generates and/or inhibit or reduce CaMKII, IP3R (including but not limited to IP3R1, IP3R2, IP3R3), Calcineurin, p38, MK2/3 or any combination thereof activity and/or activation.It is used to prepare or identifies with reference to target Other methods of peptide are well known in the art.Molecular engram is for example available for the macromolecular rebuild with reference to a certain molecule Structure, such as peptide.See, for example, Kenneth J.Shea,Molecular Imprinting of Synthetic Network Polymers:The De Novo synthesis of Macromolecular Binding and Catalytic Sites, TRIP volumes 2, the 5th phase, in May, 1994；Mosbach,(1994)Trends in Biochem.Sci.,19(9)；And Wulff, G.,Polymeric Reagents and Catalysts(Ford, W.T. are compiled) ACS Symposium the 308th, 186- Page 230, American Chemical Society (1986).A kind of method for being used to prepare the structure involves the steps of： (i) around known substrate (template) the polymerizable functional monomer for showing required activity；(ii) template molecule is removed；And then (iii) polymerize the second class monomer in the room left by template with provide show it is one or more kin with template The novel molecular of required property.In addition to peptide is prepared in this way, other binding molecules can be also prepared, such as polysaccharide, nucleosides, medicine Object, nucleoprotein, lipoprotein, carbohydrate, glycoprotein, steroids, lipid and other bioactive substances.This method is applicable in It is a variety of more more stable than their natural counterpart (because they are the free radical polymerization systems by functional monomer extensively in design It is standby) biosimulation object, so as to generate the compound with not biodegradable skeleton.For designing the other of the molecule Method includes such as drug design based on structure-activity relationship, needs to synthesize and assess many compounds and carries out molecule Modeling.
Screening test.Test compound or medicament can be identified by the measure of following two types：(a) cell base is surveyed It is fixed；Or (b) cell-less measurement.It can be the binding assay for including directly or indirectly measuring the combination of test compound to measure.It measures Or the determination of activity of the activity including directly or indirectly measuring compound.It measures or including directly or indirectly measurement MRNA nucleotide sequences are measured by the expression of the expression of the protein of target gene coding.Various screening test can with including measurement Compound is tested to combine the in vivoassay of the raised effect of symptom or hepatic glucose of metabolic disorder or coronary artery disease.Body Interior measure also is included in assessment in known mammalian animal model and tests compound to metabolic disorder or coronary artery disease or liver The raised effect of glucose.
Also the survey of the test compound for the activity screened combining target protein or adjust target protein can be carried out It is fixed.By any suitable means such as test compound can be obtained from Conventional compounds library.It can be by making test compound with putting Injectivity isotope or enzyme mark are coupled so that the combination of the cell of test compound and expression target protein can be multiple by detecting The compound measurement of the mark in object is closed to realize the ability for the protein for measuring test compound combination film combining form. For example, test compound can use3H、14C、35S or125I is directly or indirectly marked, and then can be emitted by direct Count radioactivity Or radio isotope is detected by scinticounting.Alternatively, test compound can use such as horseradish peroxidase, alkaline phosphorus Sour enzyme or luciferase carry out enzyme mark, and are marked by measuring appropriate substrate to the detection enzyme that is converted of product.
The target of target protein or target protein can be fixed to contribute to the complex for making one or two kinds of protein Formula is separated with non-complex form.Test compound and target protein can be realized in suitable for any container containing reactant (such as CaMKII, IP3R (including but not limited to IP3R1, IP3R2, IP3R3), calcineurin, p38 or MK2/3 or its change Body) combination or exist and there is no test compound in the case of, the interaction of target protein and target molecule.This The example of class container includes microtiter plate, test tube and microcentrifugal tube.In one embodiment, it is possible to provide addition allows one Kind or two kinds of protein are incorporated into the fusion protein of the structural domain of matrix (for example, glutathione-S-transferase (GST) merges egg White or GST fusion protein is adsorbable in adsorbed onto glutathione agarose pearl grain (Sigma Chemical； St.Louis, Mo.) or glutathione derived from microtiter plate).
Target protein or its variant can also be fixed by being incorporated into solid carrier.The non-limit of suitable solid carrier Property example processed include glass or plastic slide, tissue culturing plate, microtiter well, pipe, silicon chip or as bead (including but it is unlimited In latex, polystyrene or glass beads) particle.Any method as known in the art can be used in making polypeptide (or multinuclear Thuja acid) or its variant or test compound be connected to solid carrier, include the use of covalent and non-covalent bonded or Passive intake.
The screening technique of the present invention can also refer to monitoring objective protein, as CaMKII, IP3R (include but not limited to IP3R1, IP3R2, IP3R3), the expression of calcineurin, p38 or MK2/3 or its variant.It for example, can be by making cell It is contacted with test compound and measures expression of the target protein in the cell to identify the tune of the expression of target protein Control agent.By in the presence of compound is tested expression of the target protein in cell with there is no test chemical combination The expression of target protein is compared in the case of object.Test compound then relatively can be accredited as mesh based on this Mark the adjusting control agent of the expression of protein.For example, when expression of the target protein in cell is in situation existing for test compound It is lower than the statistics in the case of there is no test compound or during notable bigger, test compound is accredited as target protein and exists Stimulant/reinforcing agent of expression in cell.Alternatively, when expression of the target protein in cell is existing for test compound In the case of than the statistics in the case of there is no test compound or during notable smaller, compound is accredited as target protein and exists The inhibitor of expression in cell.Test compound is also referred to as antagonist.To measure by target gene or mRNA codings The method of expression of the protein in cell be well known in the art.
For binding assay, test compound can be to combine by the polypeptide of target gene coding or its variant and occupy its The small molecule of binding site.This can make ligand binding site that can not be reached by substrate so that normal biological activity is prevented.This The example of micromolecular includes but not limited to small peptide or peptide sample molecule.In binding assay, compound is tested or by target gene The polypeptide of coding can include detectable label, such as fluorescence, radio isotope, chemiluminescence or enzyme mark (such as alkaline phosphatase Enzyme, horseradish peroxidase or luciferase).It can then be emitted by direct Count radioactivity, by scinticounting or passed through Measure be converted definite test compound to be incorporated into polypeptide by target gene coding of the appropriate substrate to detectable product Detection.
Real-time Biomolecular transactional analysis (BIA) can also be used and measure test compound combining target albumen to realize Matter ability [McConnell etc., 1992, Science257,1906-1912；Sjolander,Urbaniczky,1991, Anal.Chem.63,2338-2345].BIA is a kind of for the in situ study life in the case where not marking any interactant Technology (such as the BIA-core of object specificity interactionTM).The variation of optical phenomenon surface plasma resonance (SPR) can use Make the instruction to the real time reaction between biomolecule.
It is combined or is interacted with target protein for identification and adjust the other oroteins of its activity, by target gene The polypeptide of coding can be used as bait protein according to the method implemented in this field in double cross measure or triple-crossing measure (bait protein) (Szabo etc., 1995, Curr.Opin.Struct.Biol.5,699-705；U.S. Patent number 5,283, 317).Two-hybrid system is based on the module that the most of transcription factors formed with activation domain are combined by separable DNA Property.
Functional assays.Compound can be tested and increase or decrease target protein, as CaMKII, IP3R (including but it is unlimited In IP3R1, IP3R2, IP3R3), calcineurin, p38 or MK2/3 or its variant activity ability.It can make purifying Target protein, cell membrane preparations or intact cell contact with test compound after measurement it is active.Make target protein Activity reduce at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, At least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95% or 100% test compound is reflected It is set to the potential medicament of the activity for reducing target protein, such as antagonist.Make the activity of target protein increase at least About 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least About 75%, at least about 80%, at least about 90%, at least about 95% or 100% test compound is accredited as increasing mesh Mark the potential medicament of the activity of protein, such as agonist.
Compound
The disclosure provides the method for treating and/or preventing the metabolic disorder of subject.The disclosure also provides to control It treats and/or prevents the method for suffering from the coronary artery disease of the subject of metabolic disorder.In one embodiment, illness is by fertilizer Fat disease induces.In another embodiment, illness is not induced by obesity.The disclosure also provides the liver for reducing subject The method that glucose generates.The disclosure also provide for reducing CaMKII, IP3R (include but not limited to IP3R1, IP3R2 and IP3R3), the method for the activity of calcineurin, p38 or MK2/3 or the inhibition activity.The disclosure also provides to drop Low CaMKII, IP3R (including but not limited to IP3R1, IP3R2 and IP3R3), calcineurin, the phosphoric acid of p38 or MK2/3 The method changed and/or activated.
On the one hand, the disclosure provides a kind of method for the metabolic disorder for treating or preventing subject in need, the side Method includes the compound that therapeutically effective amount is applied to the subject, so as to treat or prevent the illness.
In one embodiment, compound is the inhibitor of CamKII.In another embodiment, compound is IP3R The inhibitor of (including but not limited to IP3R1, IP3R2 and IP3R3).In one embodiment, compound is calcium tune nerve phosphorus The inhibitor of sour enzyme.In one embodiment, compound is the inhibitor of p38.In another embodiment, compound is The inhibitor of MK2/3.In another embodiment, compound be CamKII, IP3R (include but not limited to IP3R1, IP3R2 and/ Or IP3R3), calcineurin, p38, MK2/3 or any combination thereof inhibitor.
In one embodiment, compound is the activator of CaMKII.In another embodiment, compound is IP3R The activator of (including but not limited to IP3R1, IP3R2 and IP3R3).In one embodiment, compound is calcium tune nerve phosphorus The activator of sour enzyme.In one embodiment, compound is the activator of p38.In another embodiment, compound is The activator of MK2/3.In another embodiment, compound be CamKII, IP3R (include but not limited to IP3R1, IP3R2 and/ Or IP3R3), calcineurin, p38, MK2/3 or any combination thereof activator.
As CaMKII protein, IP3R (include but not limited to IP3R1, IP3R2 and IP3R3), calcineurin, The inhibitor of p38 and/or MK2/3 or any suitable compound of activator can be used in the method for the present invention.Such chemical combination Object can be that for example small-molecule drug, peptide medicament, peptide simulate medicament, antibody (includes but not limited to monoclonal antibody, Anti-TNF-α Body, humanized antibody and fully human antibodies and antibody fragment), inhibitory RNA molecules (such as siRNA).People in the art Member is it will be appreciated that the medicament of these and other type can be used for inhibiting or reduce or increase CaMKII, IP3R (to include but not limited to IP3R1, IP3R2 and IP3R3), calcineurin, p38, MK2/3 or any combination thereof activity reduce or increase CaMKII, IP3R (include but not limited to IP3R1, IP3R2 and IP3R3), calcineurin, p38, MK2/3 or its is any The phosphorylation of combination and/or activation.
In one embodiment, the compound of the present invention is the micromolecular inhibitor of CaMKII.The inhibitor includes But it is not limited to KN-93 (N- [2- [[[3- (4- chlorphenyls) -2- acrylic] methylamino] methyl] phenyl]-N- (2- ethoxys) -4- Methoxybenzenesulphoismide)；Lavendustin C (5- ((2,5- dihydroxy benzenes methyl) amino) -2 hydroxybenzoic acid)；CK59(2-(2- Hydroxyethylamino) -6- amino cyclohexyl amino t-butyl formate -9- isopropylpurines)；KN62 (referring to Clyne etc., 1995)； DY9760e (referring to Sugimura etc., 1997)；Nocardiopsis K-252a (referring to Kase etc., 1987)；H89 dihydrochlorides (N- [2- [[3- (4- bromophenyls) -2- acrylic] amino] ethyl] -5- isoquinoline sulfonaides dihydrochloride)；PP1 analogs II1NM-PP1 (mutant kinase inhibitor II, 4- amino -1- tertiary butyls -3- (1 '-naphthyl methyl) pyrazolo [3,4-d] pyrimidine, NM)；EEF-2 kinase inhibitors NH125 (iodate 1- benzyls -3- palmityls -2-methylimidazole, iodate 1- palmityl -3- benzene Methyl -2-methylimidazole, CaM dependant kinase III inhibitor, NH125) and STO-609 (referring to Tokumitsu etc., 2002)。
In another embodiment, the compound of the present invention is that the peptide of CaMKII or peptide simulate inhibitor or activator.This Class peptide or peptide simulation inhibitor or activator include but not limited to Ant-CaMKIINtide, [Ala286]-Ca2+/ calmodulin swashs Enzyme II inhibitor 281-301, [Ala286]-Ca2+/ cam kinase II inhibitor 281-309 and CaM kinases II (290-309) Calmodulin antagonist.
In another embodiment, the compound of the present invention is the protein of CaMKII or peptide inhibitor or activator. The protein or peptide inhibitor can be recombinant protein or polypeptide, such as but be not limited to oncomodulin (oncomodulin)/ MDP14。
In another embodiment, the compound of the present invention is antibody inhibition or the activation of the component of CaMKII protein Agent.In another embodiment, the compound of the present invention is the nucleotidyl inhibitor or activator of CaMKII.Such inhibitor Including but not limited to inhibit the expression of CaMKII or its variant or siRNA, shRNA, dsRNA, Microrna, the antisense RNA of activity Molecule and ribozyme.Such nucleotidyl inhibitor may include ribonucleotide, deoxyribonucleotide or various artificial nucleosides Acid derivative.
The activity that can inhibit CaMKII or the other compounds for the activation for reducing CaMKII are further described in United States Patent (USP) Number 7,205,298 (Kuo etc.), U.S. Publication No 2004/0086973 (Duecker K.), U.S. Publication No 2010-0056494 (Winzeler etc.), U.S. Patent number 5,386,019 (Danishefsky etc.) and U.S. Patent number 6,828,327 (Kuo etc.) In, the patent is incorporated herein in its entirety by reference.
In one embodiment, the compound of the present invention is IP3R (including but not limited to IP3R1, IP3R2 and IP3R3) Micromolecular inhibitor.Such inhibitor includes but not limited to the smooth sponge from Calbiochem (EMD Millipore) Plain C, catalog number (Cat.No.) 682160.Smooth Spongistatin C is one kind from the separated oxa- quinolizine alkaloid of marine sponge and be IP3Mediation Ca2+A kind of extremely strong power of release, the permeable blocking agent (IC of reversible and film50=358nM), not with IP3Binding site is mutual Effect.It is showed more than the highly selective of the bone hypotype of 1 type ryanodine (ryanodine) receptor (RyR-1).It is also with can Inverse mode blocks the Ca that bradykinin (bradykinin) and carbaminoylcholine (carbamylcholine) induce2+From endoplasm The outflow of net storage cavern.Other micromolecular inhibitors of IP3R are included from Tocris, the boric acid amino ethoxy of Bristol, UK Diphenyl ester (also referred to as 2-APB) is (referring to Sugawara etc., 1997, EMBO J., 16:3078-88, by reference integrally simultaneously Enter herein) and caffeine.In another embodiment, the compound of the present invention is that IP3R (includes but not limited to IP3R1, IP3R2 And IP3R3) activation of small molecule agent.
In another embodiment, the compound of the present invention is IP3R (including but not limited to IP3R1, IP3R2 and IP3R3) Peptide or peptide simulation inhibitor or activator.In another embodiment, the compound of the present invention is that IP3R (includes but not limited to IP3R1, IP3R2 and IP3R3) protein or peptide inhibitor or activator.This proteinoid or peptide inhibitor can attach most importance to Histone matter or polypeptide.
In another embodiment, the compound of the present invention is IP3R (including but not limited to IP3R1, IP3R2 and IP3R3) Antibody inhibition or activator.In another embodiment, the compound of the present invention be IP3R (include but not limited to IP3R1, IP3R2 and IP3R3) nucleotidyl inhibitor or activator.Such inhibitor include but not limited to inhibit IP3R (including but not Be limited to IP3R1, IP3R2 and IP3R3) or its variant expression or activity siRNA, shRNA, dsRNA, Microrna, antisense RNA molecule and ribozyme.Such nucleotidyl inhibitor may include ribonucleotide, deoxyribonucleotide or various artificial nucleus Thuja acid derivative.
In one embodiment, the compound of the present invention is the micromolecular inhibitor of calcineurin.Such suppression Preparation includes but not limited to Elidel and tacrolimus.In another embodiment, the compound of the present invention is calcium tune nerve The activation of small molecule agent of phosphatase.
In another embodiment, the compound of the present invention is that the peptide of calcineurin or peptide simulate inhibitor or work Agent.The peptide or peptide simulation inhibitor include the calcineurin from Calbiochem (EMD Millipore) (PP2B) inhibitor cyclosporin A, catalog number (Cat.No.) 239835.The compound is that have the induced rat thymus gland of immunosupress property thin The cyclic oligopeptides of Apoptosis in born of the same parents and in mouse B cell lymphoma cell line WEH1-231.It prevents anti-in BLB cell lines IgM and the Apoptosis of ionomycin (ionomycin) induction.Cyclosporin A is compound with cyclophilin (cyclophilin) Object inhibits protein phosphatase 2B under nanomole affinity and inhibits by il-1 α (interleukin-1 α), lipopolysaccharides With the nitric oxide synthesis of TNF-α induction.It is also from embryonic stem cell inducing cardiomyocytes.
In another embodiment, the compound of the present invention be calcineurin protein or peptide inhibitor or Activator.The protein or peptide inhibitor can be recombinant protein or polypeptide.
In another embodiment, the compound of the present invention is the antibody inhibition or activator of calcineurin. In another embodiment, the compound of the present invention is the nucleotidyl inhibitor or activator of calcineurin.It is such Inhibitor include but not limited to siRNA, shRNA of the expression or activity for inhibiting calcineurin or its variant, dsRNA, Microrna, antisense rna molecule and ribozyme.Such nucleotidyl inhibitor may include ribonucleotide, deoxyribonucleotide Or various artificial nucleotide derivatives.
In one embodiment, the compound of the present invention is the micromolecular inhibitor of p38.Such inhibitor is included but not It is limited to SB202190, SB203580 and SB239063.SB203580 can be obtained from Calbiochem (EMD Millipore) Compound, catalog number (Cat.No.) 559389.It is a kind of high degree of specificity of p38MAP kinases, strength, cell-permeable, selectivity, can Inverse and ATP competitive inhibitors (IC50=34nM, the 600nM in cell in vitro).It does not significantly inhibit JNK under 100 μM With p42MAP kinases.The cellular damage and -3/7 activity of caspase (caspase) of its reduction epirubicin induction and inhibition IL-1 and TNF-α (IC50=50-100nM) are generated from the LPS person monocytic cells stimulated and person monocytic cell's cell line THP-1. It inhibits the Neurite Outgrowth (neurite outgrowth) of the Bone Morphogenetic Protein-2 induction in PC12 cells. In another embodiment, the compound of the present invention is the activation of small molecule agent of p38.
In another embodiment, the compound of the present invention is that the peptide of p38 or peptide simulate inhibitor or activator.
In another embodiment, the compound of the present invention is the protein of p38 or peptide inhibitor or activator.It is described Protein or peptide inhibitor can be recombinant protein or polypeptide.
In another embodiment, the compound of the present invention is the antibody inhibition or activator of p38.In another embodiment party In case, the compound of the present invention is the nucleotidyl inhibitor or activator of p38.The inhibitor includes but not limited to inhibit The expression of p38 or its variant or siRNA, shRNA, dsRNA, Microrna, antisense rna molecule and the ribozyme of activity.This nucleoid Glycosides acid-based inhibitor may include ribonucleotide, deoxyribonucleotide or various artificial nucleotide derivatives.
In one embodiment, the compound of the present invention is the micromolecular inhibitor of MK2/3.In another embodiment In, the compound of the present invention is the activation of small molecule agent of MK2/3.This type small molecular inhibitor of MK2/3 includes but not limited to ammonia Base cyano pyridine compound, pyrrolopyridine and carboline base MK2 inhibitor (referring to Fyhrquist etc., 2010, J.Investig, Dermatol.,130:342-344 is incorporated herein in its entirety by reference).
In another embodiment, the compound of the present invention is that the peptide of MK2/3 or peptide simulate inhibitor or activator.It is such Inhibitor peptides include but not limited to the Hsp25 kinase inhibitors that can be obtained from Calbiochem (EMD Millipore), catalog number (Cat.No.) 385880.The inhibitor is the cell-permeable peptide with 13 residues, serves as mammalian heat shock protein matter (Hsp25) kinases [the also referred to as protein kinase -2 (MAPKAP kinases -2) of the protein kinase activation of mitogen activation] is strong Power and selective depressant.Inhibition is emulative (Ki=8.1 μM) on peptide substrate and is non-emulative (Ki on ATP =134 μM).
In another embodiment, the compound of the present invention is the protein of MK2/3 or peptide inhibitor or activator.Institute It can be recombinant protein or polypeptide to state protein or peptide inhibitor.
In another embodiment, the compound of the present invention is the antibody inhibition or activator of MK2/3.In another implementation In scheme, the compound of the present invention is the nucleotidyl inhibitor or activator of MK2/3.Such inhibitor includes but not limited to press down The expression of MK2/3 or its variant processed or siRNA, shRNA, dsRNA, Microrna, antisense rna molecule and the ribozyme of activity.This Class nucleotidyl inhibitor may include ribonucleotide, deoxyribonucleotide or various artificial nucleotide derivatives.
Those skilled in the art will be appreciated that other medicaments it is applicable make CaMKII, IP3R (include but not limited to IP3R1, IP3R2 and IP3R3), calcineurin, the inhibitor or activator of p38 and/or MK2/3, and can be together with the side of the present invention Method is used together.
Pharmaceutical composition and application for therapy
The compound of the present invention can apply 1 time (for example, in the form of single injection liquid or deposit) to subject.Alternatively, The compound of the present invention can last about 2 days to about 28 days or the period of about 7 days to about 10 days applies daily to subject in need With 1 time or 2 times.The compound of the present invention also sustainable annual 1,2,3,4,5,6,7,8,9,10,11,12 time or its combination when Phase applies 1 time or 2 times daily to subject.In addition, the compound of the present invention can be co-administered with another therapeutic agent.Work as dosage During including multiple applications, the effective quantity for the compound applied to subject may include the compound after the application of entire dosage Total amount.
Any means that compound can deliver compound by the cell being suitable for subject are applied to the subject.It lifts For example, compound can be applied by being suitable for the method for transfectional cell.For eukaryocyte transfection method in the art It is well known, and including in the core or pronucleus by nucleic acid direct injection to cell；Electroporation；Liposome shifts or by lipophilicity object The transfer of matter mediation；Receptor-mediated delivery of nucleic acids；Biology impact or particle accelerate；It calcium phosphate precipitation and is mediated by viral vectors Transfection.
It can prepare and by making active ingredient in vivo in subject (such as human or animal (such as dog, cat or horse)) with medicament Site of action generate contact any means application the present invention composition with mitigate with metabolic disorder or coronary artery disease or Hepatic glucose, which generates, raises relevant symptom.They can individually be controlled by any conventional means that can be used together with drug It treats active ingredient form or is applied with the combining form of therapeutic activity ingredient.They can be administered alone, but usually with being based on Selected administration method and the pharmaceutical carrier of standard pharmaceutical practice selection are applied together.
The compound of the present invention can effectively treat or prevent metabolic disorder or coronary artery disease or reduce hepatic glucose The amount of generation is applied to subject.Those skilled in the art can be easy to considering that compound is that prevention property is used or therapeutic made With it is lower and in the case where considering other factors determine what by be treat to subject apply the compound of the present invention effective quantity, institute State age, weight and the gender of other factors such as subject；The other medicines that subject can take；What subject can have Any allergy or contraindication etc..For example, effective quantity can be external or internal including analyzing by those of skill in the art using known procedure Definite titration curve is determined.In addition, those skilled in the art can be exploratory by being carried out in suitable animal model species It tests and is scaled up depending on subject weight etc. or reduces dosage to determine effective dose.It also can be by being used as and subject Clinical test is carried out in the individual of identical species, such as is started under low dosage and is gradually increased dosage and monitor to metabolic disease The effect of disease or coronary artery disease determines effective quantity.Also can be determined by those skilled in the art under without excessively experiment Appropriate dosage regimen is to determine for example with single dose or apply medicament with multidose, and in the case of multidose, Determine the significant interval between dosage.
The treatment for treating or preventing metabolic disorder or coronary artery disease or reducing the compound of hepatic glucose generation is effective Dosage may depend on as many factors known to those of ordinary skill in the art.The dosage of compound can be for example regarding treated tested The identity of person or sample, size and symptom are further regarded using the approach used in composition and when applicable, and practitioner needs Compound acts on target target and changes.This tittle can be easy to be determined by those of skill in the art.This tittle includes for example every Mg or microgram (μ g) amount of kilogram (kg) subject weight, such as from about 0.25mg/kg, 0.5mg/kg, about 1mg/kg, about 2mg/kg, About 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg or about 10mg/kg or About 0.25mg/kg is to about 0.5mg/kg, 0.5mg/kg to 1mg/kg, 1mg/kg to 2mg/kg, 2mg/kg to 3mg/kg, 3mg/kg To 4mg/kg, 4mg/kg to 5mg/kg, 5mg/kg to 6mg/kg, 6mg/kg to 7mg/kg, 7mg/kg to 8mg/kg, 8mg/kg extremely 9mg/kg or 9mg/kg is to any scope between 10mg/kg or between.This tittle also includes the unit dose of compound, For example, at least about 0.5mg, 1mg, 2mg, 3mg, 5mg, 10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg、110mg、120mg、130mg、140mg、150mg、160mg、170mg、180mg、190mg、200mg、225mg、 250mg、275mg、300mg、325mg、350mg、375mg、400mg、425mg、450mg、475mg、500mg、525mg、 550mg, 575mg, 600mg, 625mg, 650mg, 675mg, 700mg, 750mg, 800mg, 850mg, 900mg or more than 900mg. Any treatment use as described herein is all applied to need any subject of the therapy, including such as mammal, such as Dog, cat, cow, horse, rabbit, monkey, pig, sheep, goat or people.
Pharmaceutical composition used according to the invention can be in a usual manner using one or more physiologically acceptable carriers Or excipient.The therapeutic combination of the present invention can be prepared for a variety of administration method, applied including systemic and surface or part With.Technology and preparation can be generally found inRemmington’s Pharmaceutical Sciences,Meade Publishing Co., in Easton, Pa (the 20th edition, 2000), the entire disclosure of the books is hereby incorporated herein by.For Systemic administration, applicable is injection, including in intramuscular, intravenous, peritonaeum and being subcutaneously injected.It is of the invention for injection Therapeutic combination can be prepared in liquid solution, such as in PHYSIOLOGICALLY COMPATIBLE buffer solution, such as Hank's solution (Hank's Solution) or in Ringer's solution prepare.In addition, therapeutic combination can be prepared and before the use at once again in solid form Dissolving is suspended.Also lyophilized form is included.The pharmaceutical composition of the present invention is characterized in that at least sterile and apyrogeneity.These medicines Object preparation includes the preparation used for people and veterinary science.
According to the present invention, pharmaceutically acceptable carrier may include can be compatible with medicament administration any and all solvent, Decentralized medium, coating, antibacterial agent and antifungal agent, isotonic agent and absorption delaying agent etc..For the such of pharmaceutically active substance The use of medium and reagent is well known in the art.Any conventional media or medicine that can be compatible with reactive compound can be used Agent.Complementarity reactive compound also may be incorporated into composition.
The present invention also provides the pharmaceutically acceptable carrier for including being packed together with operation instructions and uses the present invention Screening test identification compound kit.
Pharmaceutical composition containing the compound of the present invention can be reached together with pharmaceutically acceptable carrier application Any therapeutic effect discussed herein.Such pharmaceutical composition can be included for example for the polypeptide encoded by target gene or its change The agonist and antagonist of the antibody of body or the polypeptide encoded by target gene.Composition can individually or at least one can in appoint The other reagents applied in what sterile, biocompatible pharmaceutical carrier such as stabilize compound combination application, the carrier Including but not limited to physiological saline, buffer saline, dextrose and water.Composition can individually or with other medicaments, drug or Hormone combinations are applied to patient.
The pharmaceutical composition of the present invention is prepared with can be compatible with its predetermined administration method.The example of administration method includes stomach It is parenteral, for example, intravenously, intradermal, subcutaneous, oral (for example, sucking), percutaneous (part), transmucosal and per rectum apply.With It may include following components in the solution or suspension of parenteral, intradermal or subcutaneous administration：Sterile diluent, as water for injection, Normal saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetics；Antibacterial agent, such as benzyl alcohol Or methyl p-hydroxybenzoate；Antioxidant, such as ascorbic acid or sodium hydrogensulfite；Chelating agent, such as ethylenediamine tetra-acetic acid；Buffering Agent, such as acetate, citrate or phosphate；With for adjusting the reagent of tension, such as sodium chloride or dextrose.Usable acid or Alkali, such as hydrochloric acid or sodium hydroxide adjustment pH.Parenteral preparation can be enclosed in the ampoule as made from glass or plastics, disposable injection In device or multiple dose vials.
Include aseptic aqueous solution (in the case of water-soluble) or dispersion liquid suitable for the pharmaceutical composition that injectable uses With the aseptic powdery for extemporaneous preparation of sterile Injectable solution or dispersion liquid.For intravenously applying, suitable carrier includes Physiological saline, bacteriostatic water, Cremophor EMTM(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS) (PBS).Institute Under there is something special, composition must be sterile and to there is for easy syringeability should be fluid.It is under conditions of manufacture and storage It must stablize and must be prevented from the contamination of the microorganism such as bacterium and fungi.Carrier can be to contain such as water, ethyl alcohol, pharmacy Upper acceptable polyalcohol (such as glycerine, propylene glycol, liquid macrogol) and its solvent or decentralized medium of suitable mixture.It is suitable When mobility can for example by using coating (such as lecithin (lecithin)), in dispersion liquid in the case of by maintenance needed for grain It spends and is maintained by using surfactant.Prevent microbial action (such as right by various antibacteriums and antifungal agent Hydroxybenzoate, methaform, phenol, ascorbic acid, thimerosal (thimerosal) etc.) reach.In many cases, can fit It is to include isotonic agent in the composition, such as sugar, polyalcohol (such as mannitol, sorbierite), sodium chloride.Extend injectable The absorption of composition can make the reagent of absorption delay by being incorporated to, such as aluminum monostearate and gelatin are reached.
Sterile injectable solution can by with aequum by compound (such as small molecule, peptide or antibody) with (if necessary) this The combination of a kind of ingredient or ingredient that text is enumerated is collectively incorporated into appropriate solvent, prepared by subsequent filtration sterilization.In general, pass through by Reactive compound is incorporated to the sterile carrier containing basic decentralized medium and from other ingredients needed for ingredient enumerated herein In prepare dispersion liquid.In the case where being used to prepare the aseptic powdery of sterile injectable solution, the example of preparation method is applicable in It is vacuum drying and freeze-drying, generates any other required ingredient of the active ingredient Calais from its previous sterilefiltered solutions Powder.
Oral composition generally includes inert diluent or edible carrier.They can be enclosed in gelatine capsule or suppress Piece agent.For the purpose of oral therapeutic application, reactive compound can merge with excipient and with tablet, pastille or capsule shape Formula uses.Fluid carrier can also be used to prepare the oral composition as mouth-wash, wherein the compound warp in fluid carrier Mouth is applied and gargles and spue or swallow.
It can be included in the part of the adhesive and/or adjuvant substance of pharmaceutically compatible as composition.Tablet, pill, The compound containing any following component or with similarity such as capsule, pastille：Adhesive, such as microcrystalline cellulose, yellow alpine yarrow Glue or gelatin；Excipient, such as starch or lactose；Disintegrant, as alginic acid, sodium starch glycollate (Primogel) or corn form sediment Powder；Lubricant, such as magnesium stearate or sterotes；Glidant, such as colloidal silicon dioxide；Sweetener, such as sucrose or saccharin；Or it adjusts Taste agent, such as peppermint, gaultherolin or orange flavoring.
Systemic administration can also be carried out by transmucosal or percutaneous means.For transmucosal or transdermal administration, it is suitable for The bleeding agent of barrier to be infiltrated is used in preparation.Such bleeding agent is commonly known in the art, and for example for warp Mucosal administration, including detergent, bile salt and fusidinic acid (fusidic acid) derivative.It can be by using nasal spray agent Or suppository realizes mucosal administration.For transdermal administration, reactive compound is configured to such as ointment generally known in the art Agent, ointment, gelling agent or creme.In some embodiments, compound can be reached by slow release of active compounds The transdermal delivery system application of Transdermal absorption.The percutaneous of active factors that penetration enhancers can be used for promoting to adjust in medium is oozed Thoroughly.Transdermal patch is described in such as U.S. Patent number 5,407,713；U.S. Patent number 5,352,456；U.S. Patent number 5,332, 213；U.S. Patent number 5,336,168；U.S. Patent number 5,290,561；U.S. Patent number 5,254,346；U.S. Patent number 5, 164,189；U.S. Patent number 5,163,899；U.S. Patent number 5,088,977；U.S. Patent number 5,087,240；United States Patent (USP) Numbers 5,008,110；In U.S. Patent number 4,921,475.
The application of compound is not limited to single channel, but can cover and be administered through a variety of ways.For example, Carry out through a variety of ways it is exemplary application especially include intradermal apply with intramuscular application or intradermal apply with it is subcutaneous The combination of application.A variety of applications can be carried out sequentially or concurrently.The other medication patterns carried out through a variety of ways will be skilled skill Art personnel show and are apparent from.
The compound of the present invention can be configured to composition to treat to subject's application and/or prevent metabolic disorder or hat Coronary disease reduces hepatic glucose generation.Such composition can include the compound of the present invention and one or more pharmaceutically It acceptable diluent and/or carrier and chooses any one kind of them or the mixture of a variety of other pharmaceutically acceptable additives.Medicine Acceptable diluent and/or carrier and any other additive can be compatible with other ingredients of composition and having on Evil must be " acceptable " in the sense that composition is by the subject of application.Those skilled in the art can be for example using such as Remington ' s Pharmaceutical Sciences, the 18th edition, (Mack Publishing Company:Easton, Pa., 1990), the 1635-36 pages) standard textbook in introduction, and be easy under route of delivery selected by consideration by the present invention Compound be configured to be suitable for such as people experimenter subject application composition.
The example of workable diluent and/or carrier and/or other additives include but not limited to water, glycol, oil, It is alcohol, aqueous solvent, organic solvent, DMSO, normal saline solution, physiological buffered solution, peptide carrier, starch, sugar, preservative, anti- Oxidant, colorant, pH buffer, granulating agent, lubricant, adhesive, disintegrant, emulsifier, adhesive, excipient, filling Agent, glidant, solubilizer, stabilizer, surfactant, suspension, tonicity agents, viscosity-modifying agent, carboxymethyl cellulose, crystallization Cellulose, glycerine, Arabic gum, lactose, magnesium stearate, methylcellulose, powder, physiological saline, sodium alginate.It can consider The property (such as dissolubility and stability of activating agent) of activating agent used, route of delivery (such as oral, parenteral etc.), medicament Whether (as from control release capsule delivered) will be delivered after prolonging period, whether medicament will co-administer with other medicaments and each The combination of diluent and/or carrier and/or other additives used in changing under kind other factors.Those skilled in the art will It is readily able to without preparing compound under excessively experiment for required purposes.
The compound of the present invention can be by allowing medicament to apply any appropriate methodology of its effect to subject in vivo It is applied to subject.For example, composition can be applied by known procedure to subject, described program include but not limited to pass through through Mouthful application, sublingual or buccal application, parenteral administration, transdermal administration, by sucking, passing through nasal delivery there, Via vagina, per rectum And intramuscular.The compound of the present invention can it is parenteral or by fascia, intracapsular, intracutaneous, subcutaneous, intradermal, intrathecal, muscle In interior, peritonaeum, breastbone is interior, intravascular, intravenous, parenchymal tissue or sublingual are applied.Delivering can by injecting, being transfused, Catheter delivery or some other means, are such as carried out by tablet or spray.In one embodiment, by directly delivering To the mode of heart tissue, such as in the heart of insertion experimenter or by way of neighbouring conduit or by using can make The delivery vehicle of drug targeting heart is applied the compound of the present invention to subject.For example, the compound of the present invention can sew It is applied together in the medicament (such as antibody or antibody fragment) for targeting heart or together with the medicament.In one embodiment, By way of being directly delivered to target muscle tissue, such as pass through in the target muscles of the insertion experimenter or side of neighbouring conduit Formula or by using can make drug targeting muscle delivery vehicle (such as antibody or antibody fragment) come to subject application this The compound of invention.
For oral administration, the preparation of the compound of the present invention can be rendered as capsule, tablet, powder, particle or suspension Or solution.Preparation can contain conventional additives, such as lactose, mannitol, cornstarch or potato starch, adhesive, crystallization Cellulose, cellulose derivative, Arabic gum, cornstarch, gelatin, disintegrant, potato starch, sodium carboxymethylcellulose, Calcium phosphate dibasic anhydrous or sodium starch glycollate, lubricant and/or magnesium stearate.
For parenteral administration (that is, passing through the approach application in addition to alimentary canal), the compound of the present invention can with by The aseptic aqueous solution that the blood of examination person is isotonic combines.The preparation can be prepared in the following manner：Active ingredient is dissolved in and is contained Have in PHYSIOLOGICALLY COMPATIBLE substance (such as sodium chloride, glycine) and water with buffer pH that can be compatible with physiological condition to generate water Solution then makes the solution sterile.Preparation may be provided in unit dose or multi-dose container, in sealed ampoule or bottle. Preparation can be by injecting, being transfused or other means as known in the art deliver.
For transdermal administration, the compound of the present invention can be with increasing permeability and permission of the skin to the compound of the present invention Compound is through skin and enters skin penetration enhancer (such as propylene glycol, polyethylene glycol, isopropanol, ethyl alcohol, the oil in blood flow Acid, N-Methyl pyrrolidone etc.) combination.The compound of the present invention also can further with polymeric material (such as ethyl cellulose, hydroxypropyl Base cellulose, Ethylene/vinyl acetate, polyvinylpyrrolidone etc.) be combined to provide composition in gel form, will described in Composition is dissolved in the solvent such as dichloromethane, is evaporated with viscosity needed for acquisition and is then coated in substrate material providing patch Piece.
In some embodiments, provided with unit dosage forms (such as tablet, capsule or single dose is injected or infusion bottle) The compound of the present invention.
Combination treatment
The method according to the invention, the compound of the present invention can with single medicine type or with one or more other medicaments It combines and is applied to subject.In one embodiment, the compound of the present invention is applied to subject with single medicine type. In one embodiment, independent the compound of the present invention is applied to subject.In one embodiment, the compound of the present invention It is applied with one or more other pharmaceutical agent combinations to subject.
In certain embodiments, the compound of the present invention can with for treating or preventing metabolic disorder or coronary artery disease Other pharmaceutical agent combinations of the hepatic glucose of disease or reduction subject use.In certain embodiments, the compound of the present invention can Other pharmaceutical agent combinations with being not used in the hepatic glucose for treating or preventing metabolic disorder or coronary artery disease or reducing subject It uses.In one embodiment, the compound of the present invention can be as the same drug containing one or more other activating agents A part for composition or preparation is delivered to subject.In another embodiment, the compound of the present invention can only contain and be somebody's turn to do The composition or dosage form of activating agent are delivered to subject, and one or more other medicaments are with independent group one or more Object or dosage form is closed to apply to subject.In one embodiment, other medicaments are not used in treatment or prevention metabolic disorder Or the hepatic glucose of coronary artery disease or reduction subject.In another embodiment, other medicaments are used to treat or prevent Metabolic disorder or coronary artery disease or the hepatic glucose for reducing subject.
The compound of the present invention and the liver for treating or preventing metabolic disorder or coronary artery disease or reduction subject Other medicaments of glucose can be applied in same time or in different time to subject.The compound of the present invention and being not used in is controlled Treatment or other medicaments of prevention metabolic disorder or coronary artery disease or reduction hepatic glucose can be in same times or when different Between to subject apply.For example, the compound of the present invention and other medicaments can each other several minutes, a few hours, a couple of days, several weeks or Application in several months, such as a part for the overall treatment regime as subject.In some embodiments, chemical combination of the invention Object can be applied before the other medicaments of application.In other embodiments, the compound of the present invention can application other medicaments it After apply.
Compound present invention as described above, including but not limited to CaMKII, IP3R (including but not limited to IP3R1, IP3R2, IP3R3), calcineurin, the inhibitor of p38 and MK2/3, can be combined with each other to treat or prevent metabolic disease Disease or coronary artery disease or the hepatic glucose for reducing subject.
In some embodiments, compared to the compound of the present invention is administered alone or is administered alone one or more other Medicament, the compound of the present invention is applied with one or more other pharmaceutical agent combinations has accumulative action.In other embodiments, Compared to the compound of the present invention is administered alone or one or more other medicaments are administered alone, the compound of the present invention with it is a kind of Or a variety of other pharmaceutical agent combinations are applied to have and acted synergistically.In some embodiments, compared to the change that the present invention is administered alone It closes object or one or more other medicaments is administered alone, the compound of the present invention can with one or more other pharmaceutical agent combinations applications Help to reduce side effect.
In some embodiments, the compound of the present invention is used as adjuvant therapy.In other embodiments, it is of the invention Compound is applied in combination with adjuvant therapy.
Subject
The method according to the invention, subject or patient can be suffer from or diagnose have metabolic disorder or coronary artery disease or The raised any animal of hepatic glucose.The method according to the invention, subject or patient can be to be susceptible to suffer from metabolic disorder or coronal dynamic Arteries and veins disease or hepatic glucose raise or in development metabolic disorders or coronary artery disease or the raised risk of hepatic glucose Any animal.In preferred embodiments, subject is people experimenter.In some embodiments, subject is rodent, Such as mouse.In some embodiments, subject is cow, pig, sheep, goat, cat, horse, dog and/or as domestic animal or conduct Any other animal species of raising pets.
In some embodiments, subject has suspected raises with metabolic disorder, coronary artery disease or hepatic glucose. In other embodiments, before the method according to the invention treatment, subject is just for metabolic disorder, coronary artery disease Or hepatic glucose rise is treated.In other embodiments, before the method according to the invention treatment, subject is not just It is treated for the rise of metabolic disorder, coronary artery disease or hepatic glucose.
Embodiment
Following embodiment illustrates the present invention, and is set forth helping to understand the present invention, and should not be construed in any way The scope of the present invention limited in limitation such as claim thereafter.
Embodiment 1：It is generated by calcium sensitivity enzyme CaMKII regulation and control hepatic glucoses
Hepatic glucose generates most important for glucose homeostasis.Several transcription factors and co-activating are shown Son regulates and controls this process, however, latent mechanism not yet illustrates completely.As described herein, calcium sensitivity enzyme CaMKII is in primary liver It is activated in cell by cAMP and glucagon and in vivo by glucagon and fasting in a manner of calcium and IP3R dependences. The heredity of CaMKII lacks or inhibits to block the core transposition of FoxO1, damages the glycogen point of fasting and glucagon/cAMP inductions Solution and gluconeogenesis, and reduce blood glucose level.On the contrary, gland virus expression constitutive activity CaMKII induction gluconeogenesises and decomposition of glycogen Involved in gene, glucose is stimulated to generate in vitro and in vivo, and raises blood glucose level.CaMKII lacks to glucose The containment of metabolism, which is acted through, is transduceed with composing type core FoxO1 and is abolished, so as to indicate the effect of CaMKII shortages It needs to arrange FoxO1 outside core.These results are disclosed in terms of hepatic glucose homeostasis is controlled by glucagon and fasting A kind of new calcium sensitivity molecular pathways.
Embodiment 2：It is generated by calcium sensitivity enzyme CaMKII regulation and control hepatic glucoses
Hepatic glucose generates most important for glucose homeostasis.Several transcription factors and co-activating are shown Son regulates and controls this process, however, latent mechanism not yet illustrates completely.As described herein, calcium sensitivity enzyme CaMKII is in primary liver It is activated in cell by cAMP and glucagon and in vivo by glucagon and fasting in a manner of calcium and IP3R dependences. The heredity of CaMKII lacks or inhibits to block the core transposition of FoxO1, damages the glycogen point of fasting and glucagon/cAMP inductions Solution and gluconeogenesis, and reduce blood glucose level.On the contrary, gland virus expression constitutive activity CaMKII can induce gluconeogenesis and glycogen point Gene involved in solution stimulates glucose to generate, and raises blood glucose level in vitro and in vivo.It is important that CaMKII is lacked The weary containment to glucose metabolism, which is acted through, is transduceed with composing type core FoxO1 and is abolished, so as to indicate CaMKII The effect of shortage needs to arrange FoxO1 outside core.It is as described herein that the result is shown in control liver Portugal by glucagon and fasting A kind of calcium sensitivity molecular pathways in terms of grape sugar homeostasis.
Embodiment 3：It is generated by calcium sensitivity enzyme CaMKII regulation and control hepatic glucoses
Liver is responsible for maintaining orthoglycemic major organs under the conditions of nutritional losses.During the early stage of fasting, Liver transfers glucose (Radziuk and Pye, 2001) using glycogen storage cavern.As fasting is in progress, glucose is from non-carbon hydrate The recombining of object precursor (gluconeogenesis) become hepatic glucose generation it is main facilitate process (contributor) (Klover and Mooney,2004).Glucose is generated also by the substrate flux (substrate by glycolysis, Glycogen synthesis and decomposition of glycogen Flux) regulate and control.These variations are conducted in response to direct hormone signal and quickly occurred.In addition, insulin and glucagon two It (is respectively G-6-Pase (G6pc) and phosphoenolpyruvate carboxylic that person, which can influence glycogenolytic ferment and gluconeogenesis enzyme, Kinases (Pck1)) transcription (Pilkis and Granner, 1992).During fasting, glucagon and its downstream effect The Asia of cAMP inductions such as " glucose generative nature " transcription factor of the expression of these genes of the activation of FoxO (1,3 and 4) and Crct2 Cellular localization changes (Lin and Accili, 2011).In addition, the receptor-gamma co-activating such as peroxisome proliferation activation Different co-activating of -1 α of son (PGC-1 α) and CBP are considered the different component with cAMP responses (including CREB, hepatocyte nuclear factor 4 α (HNF4 α), Sirt1 and clock gene) interaction, so as to cause gluconeogenesis gene transcription increase (Hall etc., 1995； Matsumoto etc., 2007；Puigserver etc., 2003；Rhee etc., 2003).
Calcium (Ca+2) is associated with the regulation and control of gluconeogenesis, however, latent mechanism not yet illustrate completely (Friedmann and Rasmussen,1970；Kraus-Friedmann and Feng, 1996；Marques-da-Silva etc., 1997).Evidence indicates pancreas Glucagons and cAMP change the Ca+2 flux in liver.By glucagon stimulate can cause calcium current to enter, and subsequent Ca+2 is certainly Intracellular storage cavern release, and since these change, the increase of cytosol Ca+2 concentration (Bygrave and Benedetti, 1993； Staddon and Hansford, 1989).It is worth noting that show that intracellular Ca+2 chelatings reduce glucagon induction Glucose generates (Mine etc., 1993).However, the mechanism how Ca+2 regulates and controls this phenomenon is unknown.Based on display cell These previous research of the importance of interior Ca+2, the results show activity as described herein can be in pancreas height by the increased CaMKII of Ca+2 The hepatic glucose of blood glucose element induction works in generating.
Calcium Calmodulin depedent kinase II (CaMKII) is a kind of weight of the cell Ca+2 signal transductions as in cell Want serine-threonine kinase of mediators.There are 4 kinds of genes of different CaMKII hypotypes：α, β, γ and δ.α and β hypotype masters Will be in neuron, and CaMKII γ and CaMKII δ are expressed in extensive Various Tissues.Combining, Ca+2/ calmodulins are compound After object, autophosphorylation on Thr287 generate Ca+2/ calmodulins inde-pendent activity (Couchonnal and Anderson,2008).Most of researchs on CaMKII carry out and in neuron and cardiac muscle cell in other tissues CaMKII only exist limited understanding, and specific functions of the CaMKII in metabolism is still unknown.The results show as described herein CaMKII activity is increased by cAMP and glucagon and is also responded to fasting in vivo and increase.Result explanation as described herein CaMKII works in the regulation and control of decomposition of glycogen and gluconeogenesis.Specifically, these the results shows CaMKII in vitro and There is profound influence to FoxO1 nuclear locations in a manner of regulating and controlling the expression of two kinds of key enzymes G6pc and Pck1 in vivo.
As a result
Feeding to fasting metabolism conversion causes liver CaMKII to activate
Fasting cause to have shown the cyclical level for the glucagon that can increase intracellular Ca2+ increase (Staddon and Hansford,1989).Increased CaMKII may be by fasting when intracellular Ca+2 is raised for the results show activity as described herein Activation.To test this idea, the CaMKII for glucagon the Primary mouse liver cell of various times being excited to carry out lives Property measures display CaMKII activity, and stabilization increases (Figure 1A) at any time.Diagnosis of Sghistosomiasis is carried out by using anti-pThr287-CaMKII antibody Mark confirms the state of activation of CaMKII.Consistent with kinase assays result, the phosphorylation at Thr287 is by glucagon Reason induction (Figure 1B).
To measure the effect that cytosol Ca+2 activates CaMKII, test significantly reduces the CaMKII of glucagon induction Double [2- amino-benzene oxygens] ethane-N, N, N ' of cytosol Ca+2 chelating agents 1,2- of phosphorylation, four [acetoxyl group of N '-tetraacethyl Methyl esters] (BAPTA-AM) effect (Fig. 1 C).1,4,5- inositol triphosphate receptors (IP3R) passage in endoplasmic reticulum (ER) In response to second messenger IP3 with reference to and discharge Ca2+, and play a major role in the cell in Ca+2 homeostasises.Pancreas hyperglycaemia The PKA of element induction makes IP3R phosphorylations and activation IP3R activity, and this event is important in hepatic glucose generation.To grind Study carefully the effect in the CaMKII activation that IP3R is induced in glucagon, pre-processed with the smooth Spongistatin C of IP3R inhibitor. The CaMKII phosphorylations that smooth Spongistatin C causes glucagon to induce significantly reduce, so as to illustrate IP3R in this process With key effect (Fig. 1 D).
Increase the activity of the PKA as the key enzyme involved in gluconeogenesis in response to horizontal increase of cAMP of glucagon Add.In this scenario, with the effects of the bromo- cAM processing liver cell simulation glucagons of the film of cAMP infiltration analog 8- and Phosphoric acid CaMKII is caused to dramatically increase (Fig. 1 E).Whether it is in causal correlation with PKA activation for research CaMKII phosphorylations, is adding Before glucagon liver cell is handled with PKA inhibitor H89.Inhibit the phosphoric acid that PKA significantly inhibits glucagon mediation The increase (Fig. 1 F) of CaMKII.These data support that, there are a kind of approach, wherein glucagon mediates IP3R by it The effect of intracellular Ca+2 releases promotes phosphorylation/activation of CaMKII in a manner of cAMP-PKA dependences.
To check whether CaMKII is regulated and controled in vivo by glucagon, (i.p.) glucagon in mouse peritonaeum Bolus excites.Consistent with the effect observed in the liver cell of culture, liver CaMKII phosphorylations are handled by glucagon It induces (Fig. 1 G).Glucagon dosage down to 1 μ g kg-1 can make CaMKII phosphorylations in liver.Exist to obtain IP3R Important internal evidence in the regulation and control of the CaMKII phosphorylations of glucagon mediation, mouse place in smooth Spongistatin C peritonaeums Reason 4 days.Then mouse is excited with glucagon, and measures the p-CaMKII of liver extract.As shown in figure iH, smooth sponge The CaMKII phosphorylations that plain C processing induces glucagon reduce.Then, compare in the transitional period from feeding to fasting state Between liver CaMKII phosphorylations.After fasting, liver CaMKII phosphorylations dramatically increase, and the total amount of CaMKII seems from nutrition State influences (Fig. 1 I).In addition, after feeding again, the horizontal of p-CaMKII in liver reduces (Fig. 1 J).These data show liver The activity of CaMKII is regulated and controled by nutritional status by with it in a manner that the aborning effect of hepatic glucose of fasting induction is consistent.
CaMKII promotes hepatic glucose generation, G6pc and the expression of Pck1 and FoxO1 nuclear locations in primary hepatocyte
The internal regulation and control of liver CaMKII activity are caused directly to test it in response to the feeding of fasting/again and are generated by liver cell Effect in glucose.Check that the adenovirus (gland-CA-CaMKII) with expression constitutive activity CaMKII, expression kinases are not lived Change the adenovirus (gland-K43A-CaMKII) of the CaMKII of dominant negative form or the primary hepatocyte of control gland-LacZ transductions In from the glucose of pyruvic acid and lactic acid generate.CA-CaMKII constructs have 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor at T287D, simulate that Autophosphorylation at a site and the autonomous activity of generation in the case of the Ca+2/ calmodulins there is no combination (Pfleiderer etc., 2004).Cell is checked under primary condition and after being stimulated with forskolin, the forskolin is pancreas Glucagons analogies and strength adenyl cyclase activator.Observe the basis in the cell with gland-CA-CaMKII transductions The glucose release of condition induction both increases (Fig. 2A) with the glucose release of forskolin induction.On the contrary, with gland-K43A- The glucose that CaMKII infection cells induce basic condition, which generates to generate with the glucose of forskolin induction, both reduces (figure 2A).Further to confirm these as a result, measuring the Portugal from WT mouse and in the liver cell of mouse for lacking CaMKII γ Grape sugar generates, and the CaMKII γ are the Main Subtypes for the enzyme expressed in liver cell.In the liver cell that CaMKII γ lack Glucose generation is contained (Fig. 2 B).Therefore, important work is played in the hormone regulating and controlling that glucose of the CaMKII in liver cell generates With.
CaMKII evokes the effect that hepatic glucose generates on to the rate-limiting enzyme in encoding glycogen decomposition and gluconeogenesis The research that the transcription of gene influences.For this purpose, primary hepatocyte gland-LacZ, CA-CaMKII or K43A-CaMKII transductions and survey Measure G6pc the and Pck1 gene expressions of forskolin induction.Compared to gland-LacZ, in the liver cell with gland-CA-CaMKII transductions The mRNA level in-site of G6pc and both Pck1 is significantly higher, and the transduction carried out with gland-K43A-CaMKII makes both genes MRNA level in-site reduces (Fig. 2 C).CaMKII is further supported the effect of gluconeogenesis by following observation result：Compared with WT livers Cell, G6pc the and Pck1mRNA levels of the forskolin induction in Camk2g-/- liver cell are significantly lower (Fig. 2 D).With the high blood of pancreas Sugared element processing obtains similar results (Fig. 2 E).Therefore, CaMKII is required for the expression of G6Pc and Pck1.
It is FoxO1 to induce a kind of central transcription factor involved in G6pc and Pck1, and activity is mainly by its cytoplasm Positioning regulates and controls (Greer and Brunet, 2005) compared with the variation of nuclear location.Compared with Camk2g-/- mouse, GFP marks are measured Distributions of the FoxO1 of note in the liver cell from WT mouse.It is most of under the conditions of serum starvation in WT liver cells GFP-FoxO1 is in core (Fig. 3 A).On the contrary, Camk2g-/- liver cell shows that GFP-FoxO1 is primarily located in cytosol. In addition, when liver cell is transduceed with constitutive activity CaMKII, FoxO1 becomes being mostly in core (Fig. 3 B-C).With Camk2g-/- data are consistent, and in the WT liver cells transduceed with dominant negative gland-K43A-CaMKII, FoxO1 is predominantly located at carefully In kytoplasm (Fig. 3 B).These data instruction CaMKII contributes to the FoxO1 Subcellular Localizations in serum starvation liver cell.
CaMKII γ, which lack, damages internal hepatic glucose generation and the internal hepatic glucose of constitutive activity liver CaMKII stimulations It generates
For the function affect in assessment CaMKII in vivo hepatic glucose metabolism, check WT mouse and Camk2g-/- it is small Fasting blood glucose level in mouse.It is consistent with vitro data, it observes compared with WT mouse, the blood in fasting Camk2g-/- mouse The appropriateness but statistically significant of sugar level reduce (Fig. 4 A).Compared with WT mouse, in knock-out mice, the difference of fasting glucose concentration It is different not related to Xun Huan insulin concentration increase.Mutant mice is also shown in response to pyruvic acid challenge test, plasma glucose compared with Low (Fig. 4 B).It is consistent with primary hepatocyte data, G6pc and Pck1mRNA levels and core in the liver of Camk2g-/- mouse There is reduction (Fig. 4 C-D) in FoxO1.
Further to confirm this important results, using a kind of inhibiting the adenovirus method of liver CaMKII in vivo.Phase Compared with the mouse handled with gland-LacZ, with gland-K43A-CaMKII processing C57BL/6 mouse fasting blood glucose level is caused to reduce (Fig. 5 A).It is consistent with this result of study, with liver G6pc and the Pck1mRNA expression in the mouse of K43A-CaMKII injections And core FoxO1 levels are relatively low (Fig. 5 B-C).Because FoxO1 eliminates damage both fasting gluconeogenesis and decomposition of glycogen, and CaMKII inhibits have profound influence to crucial glycogenolytic ferment G6pc, so checking that acute CaMKII inhibits to hepatic glycogen content Effect.Data show that the hepatic glycogen content in the mouse with gland-K43A-CaMKII processing increases by 60% (Fig. 5 D).To make this A little results of study are directly linked with glucagon, with injection gland-LacZ in the bolus peritonaeum of glucagon or gland- K43A-CaMKII processing mouse and then analyze liver after 30 minutes.After glucagon injection, LacZ is handled small G6pc in the liver of mouse is significantly induced, but then much smaller (Fig. 5 E) by inducing in the liver of the mouse of K43A-CaMKII processing.This Outside, the mouse of K43A-CaMKII processing has the hepatic glycogen of higher level, such as passes through periodic acid-schiff (Periodic acid- Schiff, PAS) (Fig. 5 F) observed by coloring agent.These results further confirm that CaMKII has in hepatic glucose generation Effect.
Mouse is handled by using gland-CA-CaMKII to check effects of the constitutive activity liver CaMKII in mouse.This Processing causes fasting glucose level to increase (Fig. 6 A).Feeding blood glucose level is there is also being slightly increased, but it not up to counts aobvious Work property.In addition, the mouse of overexpression CA-CaMKII shows and is applied in response to pyruvic acid that blood glucose level dramatically increases (Fig. 6 B). It is consistent with these observation results, with liver G6pc and the Pck1mRNA level and core in the mouse of gland-CA-CaMKII processing There is increase (Fig. 6 C-D) in FoxO1.The intra-body data of these combinations shows that CaMKII influences plasma glucose levels, pyruvic acid turns It is melted into the expression of glucose, core FoxO1 and hepatic glucose metabolic gene.
Glucose metabolism damage in the mouse that Camk2g-/- liver cell and CaMKII inhibit is by with composing type core FoxO1 It transduces and is saved
For importance of the verification FoxO1 in hepatic glucose metabolism is controlled by CaMKII, with containing phosphorylation deficiency group The adenovirus (Nakae etc., 2001) for being molded core FoxO1 mutant (FoxO1-ADA) transduces from Camk2g-/and the liver of-mouse is thin Born of the same parents are so that the level of core FoxO1 is similar to the level in WT liver cells.CaMKII γ shortages hold back G6pc and Pck1mRNA It makes and is abolished (Fig. 7 A) with by using gland-FoxO1-ADA transductions.It is saved with gland-FoxO1-ADA adenovirus processing mouse The damage (Fig. 7 B-D) of glucose homeostasis in the mouse of gland-K43A-CaMKII processing.In short, these results with wherein CaMKII promotes the model that hepatic glucose generates consistent by promoting the nuclear location of FoxO1.
It discusses
Glucose metabolism in liver is by insulin and the adverse effect tight control of glucagon.Many signal transductions point Son and transcription factor involved in during in the period of food is lost in the control of decomposition of glycogen and gluconeogenesis.Herein in vitro and CaMKII is added in this inventory by intra-body data, and by doing so, is provided with intracellular Ca2+ in hepatic glucose metabolism Effect molecule association.Specifically, circumferential edge shows that liver CaMKII is activated in response to fasting, so as to cause FoxO1 Core transposition and induction to decomposition of glycogen and gluconeogenesis gene.FoxO1 is in the active effects of CaMKII by following research As a result support：When in the liver cell that composing type core FoxO1 is introduced into CaMKII γ shortages, CaMKII lacks what glucose was generated Containment effect is abolished.
Glucagon-cAMP-PKA approach does not only result in CaMKII activation as shown here, and directly makes CAMP response elements on Ser133 combine (CREB) protein phosphorylation.Phosphorylation CREB transcription induction PGC1 α, the PGC1 α plays the effect (Herzig etc., 2001) for promoting G6pc1 and Pck1 transcriptions together with FoxO1.Previous research have been shown from brain The CREB of tissue also can by CaMKII on Ser133 phosphorylation in vitro (Dash etc., 1991；Sheng etc., 1991), and CaMKII The cell culture model that is generated in osteoclast of Pharmacological inhibitors in block CREB transcriptional activities (Ang etc., 2007).It does not observe To the liver cell lacked compared with CaMKII, the difference of core CREB or phosphoric acid CREB in WT liver cells, so as to exclude CaMKII Apply the possibility of its effect to hepatic glucose metabolism by modifying CREB.
FoxO1 activity mainly by include phosphorylation and acetylation posttranslational modification regulate and control (vander Horst and Burgering,2007).What is fully proved is that FoxO1 passes through Akt at Thr24, Ser256 and Ser319 by growth factor Phosphorylation is to promote to arrange outside its core.CaMKII promotes FoxO1 nuclear locations.In fact, CaMKII γ shortages do not influence these three The phosphorylation of residue.Evidence suggests the FoxO phosphorylations that other kinases by such as JNK and AMPK carry out on non-Akt sites can Its core can be essentially facilitated to retain.Therefore, the balance of FoxO activity can be by irritation phosphorylation event and inhibition phosphorylation Caused by the combination of event, and under without being bound by theory, CaMKII can be by direct zymogenesis or by influencing FoxO phosphoric acid Enzymatic activity influences this balance.The deacetylated nuclear locations (Frescas etc., 2005) that can also promote it of FoxO1, but FoxO1 Acetylation also lacks from CaMKII to be influenced.Alternatively, CaMKII γ may influence the FoxO1 in cytoplasm or core in some way Possibility involved in transposition or the expression of FoxO1 interacting molecules or activity influences input or the output mechanism of this process. Future studies, which will focus on, illustrates this mechanism.
It was found that a kind of novel molecular involved in hepatic glucose generation not only provides and the physiology for being directed to fasting hypoglycemia is prevented Imperial understanding, and the novel therapeutic for the glucose metabolism being disturbed being present in insulin resistance environment can be disclosed Target.In fact, in diabetes B, out-of-proportion hepatic glucose exports and compared with the high blood of insulin signal transduction pancreas The imbalance of sugared element facilitates hyperglycemia (Saltiel, 2001).In this scenario, future studies will solve to inhibit liver Whether CaMKII can improve obesity and the metabolic disorder of insulin resistance.
Embodiment 4：Suitable for the experimental arrangement being used together with method described herein
Reagent and antibody
Glucagon, pyruvic acid, forskolin, H89 and the bromo- cAMP of 8- come from Sigma.BAPTA-AM and anti-phosphocarnic acid egg (nucleophosmin, Np) antibody comes from Invitrogen in vain.Smooth Spongistatin C comes from EMD Chemicals.Anti- phosphoric acid Thr287CaMKII antibody comes from Imgenex and Novus；Total CaMKII and anti-FoxO1 antibody is resisted to come from Santa Cruz Biotechnology Inc, anti-beta-actin antibody come from Abcam.
Cell culture
Primary mouse liver cell is that (Matsumoto etc., 2002) as discussed previously is isolated from 8 to 12 week old mouse.Make thin Born of the same parents be subjected to staying overnight serum exhaust and be then incubated in serum free medium with forskolin (10 μm) 5 it is small when.
Measure CaMKII activity
According to manufacturer specification, CaMKII activity is measured using the CaMKII assay kits from Promega.Such as In legend after indicated processing liver cell, 50mM HEPES, the 150mM of 1% triton-X (Triton-X) are contained by being exposed to NaCl, 10mM sodium pyrophosphate, 10mM EDTA, 10mM EGTA, 1mM Na3VO4,50mM NaF, 1mMPMSF and the 5 bright peptides of μ g/ml Plain (leupeptin) 5 minutes dissolves them.Then, [γ -32P] ATP and CaMKII biotinylated peptide substrates are added to After being incubated 10 minutes in lysate and at 30 DEG C, using SAM biotins trapping film make [32P]-phosphorylated substrate with it is residual Remaining [32P] ATP is separated and quantified using scintillation counter.
Adenovirus infection
The adenovirus for encoding LacZ, CA-CaMKII, K43A-CaMKII and GFP-FoxO1 is previously described (Pfleiderer etc., 2004；Tanaka etc., 2009) and expanded by Viraquest, Inc. (North Liberty, IA). It transduces when 12 is small after inoculation primary hepatocyte.RNA and Separation of Proteins and glucose production are carried out when after transduction 24 is small It is raw.
Glucose in primary hepatocyte generates
Such as it is described progress glucose generate measure (Backs, 2010；Yoon etc., 2001).In brief, in as above institute After stating collection and culture Primary mouse liver cell, cell culture medium is converted into be supplemented with 20mM sodium lactates and 2mM pyruvic acid The DMEM (pH7.4) without glucose and phenol of sodium.After when culture 16 is small, 500 μ l culture mediums are collected, and use colorimetric Portugal Grape sugar determination kit (Abcam) measures glucose content.Then reading is made compared with the total protein concentration in whole cell lysate Number standardization.
Mouse experiment
Camk2g-/- mouse (Backs etc., 2010) is generated as discussed previously and hybridization is in C57BL6/J backgrounds.Mouse With standard diet (standard chow diet) feeding and according to 12 it is small when fight-darkness cycle supported.Pass through afterbody Intravenous injection delivering recombined adhenovirus (1.5 × 109 plaque formation unit/mouse).Use Glucose meter (One Touch Ultra, Lifescan) measurement freely obtain underwater fasting 12-14 it is small when mouse in fasting blood-glucose.In fasting 17 it is small when after, pyruvic acid tolerance test is carried out with the pyruvic acid that 2g kg-1 weight is injected in peritonaeum.It is small after subsequent 2 When measure blood glucose level.It injects to apply to mouse in daily peritonaeum by continuing to carry out for 4 days under the dosage of 10pmol g-1 Smooth Spongistatin C.
Hepatic glycogen measures
50-100mg freezing livers are homogenized in the 1mL H2O with protease and inhibitors of phosphatases.Then sample is made Sheet and KOH (1:2) mix, boil 25 minutes and washed with 70% ethyl alcohol.Make precipitation dry and be dissolved in 100 μ l H2O And according to manufacturer specification, glycogen content is assessed using glycogen assay kit (Abcam).Data represent average value ± SEM.
The PAS dyeing of mouse liver slice
By liver sample be fixed in 10% neutral buffered formalin 24 it is small when and be embedded in paraffin.It is said according to manufacturer Bright book uses the glycogen in periodic acid-schiff (PAS) coloring agent (Sigma) stained slice (5 microns).Then with hematoxylin pair It is checked than stained slice and by optical microscopy.For quantifying for PAS dyeing, random selection is from 4 different sections 5 regions and the number of PAS positive cells is counted and is expressed as the percentage of total number of cells, (Raza as discussed previously Asim etc., 2010).2 independent investigators unwitting to the identity of sample analyze.
Western blotting and quantitative RT-PCR
Using RNeasy kits (Qiagen) total serum IgE is extracted from liver cell.Use oligomerization (dT) and Superscript II (Invitrogen) synthesizes cDNA from 2 μ g total serum IgEs.Real-time qPCR analyses and Western blotting are carried out as discussed previously (Timmins etc., 2009).According to manufacturer specification, using the core extracts kit from Panomics extract from liver Core.
Statistical analysis
All results are all rendered as average value ± SEM.Using student t (student ' s t-test) is examined to calculate P values.
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Embodiment 5：The hepatic glucose that liver CaMKII is mediated in glucagon generates the effect in (HGP)
The insulin resistance and hepatic glucose and fat metabolic disturbance that obesity induces increase heart disease, cancer and other wide The risk of cloth and crushing disease.Current therapeutic option is extremely limited, and so as to have an impact, to suffer from current obesity epiphytotics The urgent of hundreds of millions overweight personnel does not meet clinical needs.After past 2 years, several discoveries (were included in the model of obesity Verification) instruction unique pharmaceutical target-be known as CaMKII liver enzyme-inhibitor can have huge valency in this microhabitat Value.Therefore, overall goal of the invention is the purpose exploitation for drug development and tests CaMKII inhibitor to improve fertilizer Fat disease, Metabolic syndrome are sought peace metabolic disorder and their consequence in diabetes B.
Show that CaMKII is activated by glucagon in primary hepatocyte and lived in vivo by glucagon and fasting Change (Fig. 1).The heredity of liver CaMKII lacks or inhibits to reduce blood glucose level, contains HGP gene G6pc and Pck1, reduces glycogen consumption It exhausts, and blocks the core transposition of HGP transcription factors FoxO1 (data on dominant negative CaMKII are shown in Fig. 5).On the contrary, Constitutive activity CaMKII induces G6pc and Pck1, and glucose is stimulated to generate, and raises blood glucose level.It is important that CaMKII is lacked The weary containment effect to glucose metabolism is abolished by composing type core FoxO1, so as to indicate that the effect that CaMKII lacks is needed outside core Arrange FoxO1 (Fig. 7).These results disclose a kind of novel molecular approach regulated and controled by CaMKII in terms of HGP is controlled.
Obesity is checked, because glucagon, which is more than the imbalance of insulin signal transduction, can facilitate the height in obesity Blood glucose and insulin resistance.In the liver of obesity (DIO) mouse of (ob/ob) mouse and meals induction of leptin (leptin) shortage In, CaMKII is activated, but (Fig. 8 A) is not activated in the liver of modest mouse.In the liver of obese people, CaMKII is also activated, But (Fig. 8 B) is not activated in lath.The liver CaMKII that most importantly heredity or pharmacology inhibit in obesity mice reduces empty Abdomen blood glucose；Improve insulin resistance；Reduce the expression of the key gene involved in HGP；Mitigate fatty liver and inflammation and improve blood Fat is abnormal (Fig. 9-10).These data supportive treatments target the hyperglycaemia and insulin that CaMKII will improve in obese subjects The concept of resistance.
CaMKII γ antisense oligonucleotides (ASO) will be tested in the mouse model of obesity
It will be tested for the ASO of Camk2g in the mouse model of obesity.Isis have control ASO and 4 kinds not With Camk2g ASO, will be used to handle DIO obesity mices (continuing the weekly interior injection of peritonaeum in 6 weeks).Liver and its will be measured It organize in Camk2gmRNA level and overall tolerability will be assessed, including liver weight, liver functional test and to fasting Tolerance.The effect of parameter that testing improvement is following：Fasting and feeding plasma glucose and insulin；HGP gene expressions and FoxO1 nuclear locations；Pass through hyperinsulinemia euglycemic clamp (hyperinsulinemic euglycemic clamp) Study the HGP obtained and periphery insulin sensitivity；Fatty liver；And plasma triglyceride and aliphatic acid.
Screening and the new chemical inhibitor of test CaMKII：
The high throughput fluorescence based assays to CaMKII activity will be used to screen " can drug " chemistry library, the measure Dependent on the fluorescent emission when inactivating enzyme than variation.By definite structure-activity relation and existing and commercially available use will be made It is optimized in the efficiency and specificity of external and preclinical study CaMKII inhibitor.Most promising target (hit) will connect It and postsearch screening is carried out in the model of insulin resistance primary hepatocyte, this is related to simulate seen in the liver in obesity mice Glucose generate and insulin resistance increased mode saturated fat acid treatment cell.Using this model, will check CaMKII, glucose generate and the most potent inhibitor of insulin resistance.By according to above for ASO general introduction strategy to gram Most promising drug prepared by quantity carries out internal test, wherein the inhibition in the activity and non-liver tissue to monitoring CaMKII Consequence is maintained vigilance.The orally delivering simplification of available drug and the delivering to liver will be assessed, i.e., delivered by portal circulation.Such as Fruit, which changes drug, becomes necessary, then can change drug to realize oral availability.
Embodiment 6
Increased two principal elements of risk for promoting coronary artery disease (CAD) in obesity/insulin resistance are spots Actuate arteries and veins atherogenic process and systemic risk factors (particularly liver source property dyslipidemia) in block itself increase.People's diabetes Property patch be characterized in that promoting the necrosis area of inflammation, plaque rupture and acute CAD especially larger.Patch necrosis is due to dead In the defective environment of cell clearance macrophage () apoptosis and develop.It will check the extended endoplasm in advanced lesions Net (ER) stress triggerDead and patch necrosis institute foundation mechanism.These processes are defective in insulin signal transductionIn be extended.Extended ER stress promote cytoplasm calcium (Ca2+) raise, the calcium by activate Ca2+/calmodulin according to Property protein kinase II- γ (CaMKII γ) is relied to trigger Apoptosis.Missing or the CaMKII γ inhibited in obesity mice can Being protected from hyperinsulinemia, gluconeogenesis, dyslipidemia, fatty liver and ER stress.These recent studies on results indicate a kind of enzyme There can be key effect (Figure 11) to the two kinds of supplement processes for the CAD for promoting insulin resistance subject.Therefore, CaMKII γ shortages can be reducedAdvanced lesions in the mouse model of insulin resistanceApoptosis and patch necrosis and Liver CaMKII γ lack the Atherogenic metabolic disorder that will improve in obesity mice.
CaMKII γ lack the advanced lesions that will be reduced in insulin resistance mouse Apoptosis and patch Necrosis
Vitro data show CaMKII γ coordinate ER stressIn many key cells apoptosis pathway.PPG data is shown Show insulin resistanceIn Ca2+ metabolism multilated and CaMKII γ be activated.Therefore, CaMKII γ can ER stress Insulin resistanceApoptosis in play special important function.It will first determine from deficiencyInsulin signal transduction Two conceptions of species verification model Insr-/- and ob/ob it is separatedIn siRNA mediation Camk2g silences whether can contain The Apoptosis of the ER stress-induceds of very high degree in these cells, and then probe into molecule/cell mechanism of protection.It will survey The p-CaMKII as the measurement of CaMKII activation is measured to determine compared with the earlier stage atherosclerosis in people and mouse Lesion, it is late neuropathic in stage atherosclerotic lesionIn it is whether higher.To test reason, will come from Camg2gfl/flLysmcre+/- Insr-/- mouse and from WT mouse and from shortageCaMKII γ or insulin The bone marrow cell of the mouse of receptor is migrated to respectively in Ldlr-/- mouse, and is then seated in Western diet (Western Diet on).The advanced lesions of high level in Insr-/- → Ldlr-/- mouseApoptosis and patch necrosis by becauseCaMKII lacks and significantly improves.
Liver CaMKII γ lack the metabolic disorder for improving obesity and liver specificity CaMKII γ shortages are fat by containment The mechanism that the atherosclerosis of mouse is relied on.
Camk2gfl/fl X alpha1-antitrypsin-Cre mouse will be used to whether test liver specificity CaMKII γ shortages The glucose in obesity can be improved, lipid and lipoprotein is metabolized.By test obesity by being related to the sub- CHOP's of ER stress effects The idea of mechanism activation liver CaMKII γ.It is a kind of to the beneficial effect of glucose and lipid-metabolism that liver CaMKII γ shortages will be probed into FoxO1 is arranged outside potential mechanism, i.e. core and inhibits hepatic glucose generation/gluconeogenesis.It will detect compared with lath subject, come from CaMKII activation and related mechanism in the liver sample of fat people experimenter.LysMCre models will be used to study liverIn CaMKII γ lack whether by contain ER stress and/or inflammation work and whether it can also promote in obesity Liver mediation metabolic disorder containment.Following item will be tested：(a) liver in the Ldlr- of Western diet feeding/- mouse Whether CaMKII γ lack forms containment atheroma；(b) combination liver andWhether CaMKII γ shortages will be right The atherosclerosis in all stages has notable beneficial effect.
Embodiment 7：Patch necrosis
2-3% causing the lesion of acute CAD not by their large-size and by there are patch necrosis distinguish 1,2, institute It states patch necrosis and promotes plaque rupture, acute tube chamber thrombosis and histic infarct (3).Patch necrosis byApoptosis with ExtremelyRemoving or the combination of " born of the same parents bury (efferocytosis) " defective (so as to cause downright bad after Apoptosis) cause (4-6).This concept is especially important in terms of how consideration diabetes and insulin resistance promote CAD, because diabetes are tested Advanced atherosclerosis lesion in person is characterized in that when compared to the similarly sized lesion from non diabetic individuals When, necrotic cores are especially big (7-12).The perspective study of subject to suffering from CAD finds only diabetes and age and necrosis Kernel size positive correlation (12).These data are proposed on advanced lesionsWhether Apoptosis increases in diabetes environment The problem of strong.
As advanced lesions The extended ER of the mechanism of Apoptosis stress
Mechanism and intra-body data support that extended ER stress be late neuropathicHave in Apoptosis and patch necrosis It acts on { 4997,5081 }.ER stress-inducedsApoptosis depends primarily upon so-called unfolded protein matter response (UPR) CHOP branches (15,16).In human coronary artery, in CHOP expression, Apoptosis and there are poles between the advanced lesions stage Strong association (17).The advanced lesions progress for the atherosclerosis susceptible mouse that CHOP lacks is by containment (16), this is then by making Involved with bone marrow neoplasmsAnother small group acknowledge (18) of the effect of CHOP.Researches show that be considered mitigation ER to answer for Section 3 The atheroma protective effect (19) of sharp " chemical chaperone ".Lower means be can be used in cultureIn establish CHOP The model of cell apoptosis of induction：(a) ER stress strength derivant, be late arterial congee such as 7- ketone groups cholesterol (16,20) Most abundant oxygen sterone (oxysterol) (17) in sample hardening lesion；Or (b) more delicate ER stress add atheroma related The combination of " the 2nd target ", the target be particularly the pattern identification reached by the lipoprotein or saturated fatty acid modified by Body (PRR) activates, and the activation, which expands, promotees antiapoptotic signals conduction and containment cell survival signal transduction (21,22).2 target mesh Mark concept is also applied for advanced lesionsAlternate model, i.e., with lipoprotein source property free cholesterol (FC) load (23-26), because the excessive FC activation UPR in ER films, and the lipoprotein activation PRR modified.It is most important for this proposal It is that PPG has shown that the Apoptosis of ER stress-induceds is defective in insulin signal transductionIn significantly increase.
Rush atheroma effect of the liver in obesity and insulin resistance.CAD risk of the liver in insulin resistance It plays a major role in increase (27,28).Primary association is to reach in the following manner：VLDL apoB and triglycerides (TG) Secretion increases, this is triggered by the generation of fat again for increasing coupling with the delivering of the liver of fat-derived free fatty；Contain apoB Degradation；With enhancing apoB translations 28.The fat generation and VLDL secretions of SREBP1c mediations are by being activated by hyperinsulinemia " remnants " insulin receptor signal transduction path is stimulated (29-33).Insulin receptor-mTORC1 approach contains sorting protein (sortilin) the apoB degradations of -1 mediation.Two kinds of other potential associations between obesity, insulin resistance and metabolic disorder Be liver ER stress and inflammation.Liver ER in obesity stress can be mediated by IRE1-JNK to the IRS-1's on Ser30734 Phosphorylation contains insulin signal transduction by activating GSK-3 β, and the GSK-3 β inhibit the AKT activation of rictor mediations (35,36).Mitigate the liver ER in animal model stress operation improve insulin resistance (37,38), and the research in people is Showing that weight loss reduces liver ER stress (39).Liver ER stress promote SREBP-1c activation and steatosis (34,38,40), but To VLDL secretions, the influence of itself is still treated to probe into completely (41-43).For inflammation, cell factor can autoblood or by by Involve intrinsic Cusparia method (Kupffer) cell in the insulin resistance of obesity induction or the macrophages secrete newly raised And enter in liver (28,44,45).Inflammatory cytokine activates JNK, and the JNK is passed by destroying insulin receptor signal Lead (more than) and by activated protein kinase PKR46 and associated with insulin resistance.
CaMKII。
CaMKII is a kind of Ser/Thr kinases of Ca2+ activation, activity form be with 12-14 by 4 kinds of gene α, The equal polymer of the subunit of one of β, γ or δ 47 codings.(pay attention to：CaMKII is different from the CaMKK of activation AMPK).α and β is sub- Type is in neuron, and CaMKII γ and CaMKII δ are expressed in extensive Various Tissues.With HC expression γ forms 48. Under basic status, control domain interacts with catalyst structure domain and inhibition catalyst structure domain.When cytosol Ca2+ increases When, Ca2+- calmodulin complexes destroy this interaction, thus mitigate the self self phosphoric acid inhibited and promote Thr287 Change.This process increases the affinity that Ca2+-CaM is combined and causes Ca2+ dependent/non-dependent kinase activations.On the big of CaMKII Majority research carries out in neuron and cardiac muscle cell.In, inhibitors in vitro research has in the following have been reported Effect：Phagolysosome kills bacterium；The adenylyl enzyme activation of LPS mediations and HIF-1 α inductions；The p38 of PKR mediations Activation；The Apoptosis of autoimmunity induction；With derepressing (49-54) for inflammatory gene.On the CaMKII in liver cell Public data it is extremely limited.As illustrated in sections below, heredity targeting Camk2g is used to be shown in ER stress-induceds(48) and the liver in obesity environment drive the new effect in metabolic disorder in Apoptosis.
Effects of the CaMKII γ in atherosclerosis was previously never probed into, and it is resisted due to insulin PropertyIn calcium dynamics change caused by insulin resistance environment in importance be a kind of new concept.It is a kind of new Cre-lox models will be used to test these ideas.Effects of the liver cell CaMKII γ in the metabolic disorder of obesity can disclose Other new principles in this key area.Cre-lox models also will ideally test this idea.Finally, carried This single molecule gone out passes through to neuropathic, liver cell and may liverAct on mediation obesity and insulin Complete effect in terms of atherosclerosis in resistance environment represents a kind of novel concept with critical treatment meaning.
It will be for the CAD master after coming decade with obesity and the relevant CAD risk factor of insulin resistance epidemic disease Want driving factors (55).This result of study illustrates the new mechanism of two critical process in this field and internal consequence, One process concentrates on patchApoptosis and another process concentrate in liver the metabolic process for promoting CAD risk.It grinds Study carefully and concentrate on advanced lesions form, especially for the necrosis of the most important characteristics for a few peoples' patch for actually causing acute CAD (56).In addition, result of study includes the use of people's patch sample in various advance stages and compared with lath subject, come from The future studies that the liver sample of fat people experimenter carries out.After the research for completing to propose, resist obtaining for obesity/insulin Property subject novel therapeutic strategy, common in the strategy targeting arterial wall and liver promote atheroma signal transduction way Footpath.It is worth noting that in other schemes that CaMKII inhibitor has been considered promotion progression of disease in wherein CaMKII, dynamic It is successfully tested in object model (57,58).As described herein, the enzyme is partly inhibited to protectionHave with HC and significantly make With this further increases the feasibility of therapy.It can be by selective depression γ hypotypes and by using making drug targeting Atherosclerotic lesion and the strategy of liver obtain specific (59,60).However, it is contemplated that CaMKII inhibits in diabetes Other aspects, such as the benefit in heart failure (61), hypertension and nephrosis (62) and retinal disease (63), this is special Property may and need not.
Test Whether CaMKII γ lack will reduce insulin sensitivity and especially In-insulin resistance mouse Advanced lesions Apoptosis and patch necrosis.
In the presence of for be related to extended ER stress be combined with PRR signal transductions it is above-mentioned" the 2 of Apoptosis Target " mechanism has in vitro and in vivo evidence (22,48,64-72).One key ER stress pathways be related to CHOP mediation to ER The induction of oxidizing ferment ERO1 α, the ERO1 α then activate ER Ca2+ release channels IP3R.The Ca2+ activation CaMKII of release γ transfers and triggers downstream apoptosis approach, includes the induction to Fas death receptors of JNK- mediations；External mitochondria The release of the permeabilization and cytochrome c of film；And the ROS of induction nadph oxidase mediation, also by the upper of referred to as PKR Swim kinases amplification CHOP (Figure 12).On above-mentioned treatment potentiality, the part of CaMKII γ inhibits to be enough to protect(figure 13).It is important that ER stress CaMKII γ lack mouse and protected and exempt from peritonaeum and spleenAnd in renal epithelial cell Apoptosis in vivo.However,- CaMKII γ late atherosclerotic lesionsApoptosis and patch necrosis In effect it is unknown.
These concepts are applied in insulin resistance environmentThe main target of PPG is become.By at least The Apoptosis of 3 kinds of mechanism enhancing ER stress-induceds：(a) scavenger receptor (74) is raised；(b) containment AKT and NF- κ B cells are deposited Approach (66) living；Lower ER Ca2+ pump SERCA75 (c).Most importantly advanced lesionsApoptosis and patch are bad Extremely whereinIncrease by 76 in lesion with deficiency insulin signal transduction, this very big necrosis with people's diabetes lesion Core is consistent.It is another that researches show that work asWhen being Insr-/- or Irs2-/- 77, the high cholesterol/cholate of feeding The lesion area of the blending heredity background Apoe-/- mouse of (" Paigen ") meals moderately reduces.This can be with pro-inflammatory Anti-inflammatory effect in Paigen Hydrocarbon Insecticides in Dietary Environment is related (77-79), consistent with more than NF- κ B researchs (66).It does not wrap most importantly Include advanced lesionsApoptosis and the crucial terminal of patch necrosis, the key terminal are the focuses of PPG.
Under without being bound by theory, this 3 kinds of mechanism and ER stress/2 target models of PRR between associate and be easy to be based on These mechanism and new data (Figure 14) and it is aobvious and be apparent from：(a) in insulin resistanceIn, ERK1/2-SERCA approach is suppressed System, this increases ER Ca2+ and discharges into cytosol (75).It is as described herein that the result is shown in Insr-/- and ob/obIn, The p-CaMKII of label as CaMKII activation increases, and the WT inhibited in ERKIn it is consistent with more than mechanism (75) (Figure 15)；(b) inhibit SERCA can extend ER stress (80), and CaMKII activation by feedover cycle expand ER (stress scheme 16)-it is all with from ob/ob mouse fresh separatedsIn the increased results of study of CHOP (68) it is consistent.
Mechanism Study.
By compared with insulin resistance ER stress, measure the CaMKII activity in control.It, will to widen importance Use separate sources, the derivant of insulin resistance, ER stress activator and the method for silence CaMKII γ. / the peritonaeum of-mouse will be used from Insr-(76), this is to be shown based on insulin receptor as caused by hyperinsulinemia It writes and lowers (81-84), including lowering (85,86) to the notable of the monocyte from insulin resistance people.ThesevWTWill with following atheroma correlation ER stress activator (13) excite：FC totes；7- ketone group cholesterol；Or low dosage ER stress add PRR activators, such as peroxynitrite donor SIN-1 to add oxidized phospholipids (oxPL) (72).It will test as CAD Risk factors (87-91), oxPL in people carrier (92) and ER stressIn Apoptosis strength stimulant (72) lipoprotein Lp (a).It will be surveyed by pThr287 Western blottings (Figure 15) or by direct CaMKII kinase assays (48) Determine CaMKII activation.
ER stress insulin resistanceIn the degree of Apoptosis be made of two components：" basic " (that is, ER should Sharp WTSeen in degree) plus because insulin resistance generate increment, wherein each to final cell Apoptosis response tribute It offers about 50% (76).In ob/obIn, CaMKII inhibition in part causes the basic component of the Apoptosis of ER stress-induceds The incremental portion for reducing about 40% and ob/ob inductions inhibits about 65%, i.e. total cell apoptosis is existed by the degree that inhibitor reduces ob/ob In be more than WTER stressIn (in WT 5.0 ± 0.4 → 3.1 ± 0.2% compared in ob/ob 11.9 ± 1.1 → 5.6 ± 0.5%).These experiments will use (a) ob/oIn Camk2g siRNA；(b) ob/ob is compared with ob/ obCamk2g-/-；From following atherosclerosis study (c)(that is, Ldlr-/- mouse with WT mouse, The bone-marrow transplantation of Camk2g KO mouse, Insr KO mouse or Camk2gInsr DKO mouse) it is repeated.It will be by determining Which effector for promoting antiapoptotic signals conduction of CAMKII γ inductions is in insulin resistanceMiddle increase and because CaMKII γ lack and reduce to detect mechanism：Fas death receptors induce；STAT1 is activated；MtDNA COⅠ gene absorbs and cell color Plain c releases；Nox2 inductions/ROS48；UPR is expanded；It is activated with PKR.Four groups—con-WT；Insulin resistance WT；con- Camk2g-/-；With insulin resistance Camk2g-/--(48,68,93,94) will be measured as follows：RT-QPCR is used for Fas MRNA and facs analysis are used for Fas cell surface expressions；Western blotting is used for p-STAT1；Rhodamine -2 dyes and mt- Pericam fluorescence (95) is used for mtDNA COⅠ gene；Western blotting is for mitochondria and cytosol cytochrome c and half Guang asparagus fern - 9 determination of activity of enzyme；RT-QPCR accumulates for Nox2mRNA and DCF dyeing for Cellular peroxide；Western blotting is used for p- PERK and CHOP；And Western blotting is used for p-PKR.Compared with sensibility ER stress, these effector terminals will be Insulin resistanceMiddle increase, and in the case of there is no CaMKII γ, insulin resistanceIn basic ER stress lure The component led will both be reduced with further increment, so as to cause Camk2g-/-Totality significantly reduce.However, pancreas Island element resistance causes the selectively increase of specific terminal or if the factor of the data instruction in addition to CaMKII γ can relate in pancreas islet In the increment of plain induction of resistance, then will conceive and perform and tested by the mechanism of data-driven.
Under without being bound by theory, insulin resistance (IR) enhancing ER stressIn the rush cell of CaMKII γ wither The effect of dying (IR → CaMKII).Positive feedback loop is often related in the study on signal transmission of apoptosis of ER stress-induceds (93) (Figure 16).Therefore, it will probe into whether CaMKII γ can feed back enhancing insulin resistance (IRCaMKII).It is if such Words, then based on previous research (66,72,74,75,75), CaMKII γ shortages can be in insulin resistanceIn have it is following Protective effect：(a) ↓ SRA and CD36, ↑ p-Akt, ↑ p-ERK, ↑ SERCA2b and ↑ p-FoxO1 (Western blotting)；(b) ↑ Serca2b, ↓ Ikbe and ↑ Bcl2 are (in insulin resistanceThe NF- κ B targets 66 of middle reduction) (RT-QPCR)；(c) ↓ ERCa2+ (fluoro- 3- thapsigargins (thapsigargin) fluorescent observation)；(gland-GFP-FoxO1 turn by ↓ core FoxO (d) It leadsIn immunofluorescence).It is nuclear locations of the FoxO1 in fasting and insulin resistance liver cell in view of CaMKII γ Institute is required, especially interested in aftermentioned measure.It is answered in short, these researchs will be provided on CaMKII γ in insulin resistance in ER SharpIn promote cells apoptosis in effect comprehensive opinion.
Human atherosclerosis.
However Reason-study will carry out (next chapters and sections) in mouse, but patch will be testedIn CaMKII γ work Change whether (a) is promoted with the patch stage and increased；And whether (b) is in deficiencyIncrease in insulin signal transduction environment Add.Fresh separated/snap frozen carotid plaques sample is divided into 3 neighbouring small sections among patch.Middle slice will be used Add in anti-p-CaMKII immunoblotting assays and by being compareed with the densitometry ratio and beta-actin of total CaMKII γ loading With quantitative.Flanking region immediately will be cut into slices with ultramicrotome and then (a) measured with Movat five colors staining reagent it is bad Unleavened dough accumulates (percentage of the quantification of gross area)；And (b) is right, p-CaMKII carry out immunostaining (Figure 17), and carry out For TUNEL (the quantification of positives of ApoptosisPercentage).Target be display p-CaMKII and necrosis area (andTUNEL the association between) is significant under P=0.05.In addition, these samples will use Virmani-AHA flows (96) 5 stages are categorized into a manner of blind：1=diffusivity intimal thickenings；2=fibrous plaques；3=thickness lid atheromas；The thin lids of 4= Atheroma；Patch is ruptured with 5=.Target is tested compared with 1+2, and in the 4+5 stages, whether p-CaMKII is enriched with.N=15 will be with 80% effect is enough to be shown between 4+5 lesions and 1+2 lesions, and at least 2 times under P=0.05 there are p-CaMKII are poor It is different.Finally, by analyze all data with obtain with there are diabetes B compared with the correction of lesion stage associate and p- CaMKII and two lesion stage and there are the positive associations between diabetes B.
Mouse atherosclerosis study.
Two targets are：(a) test CaMKII γ activation and whether its rush cells apoptosis promotes with the patch stage And increase and whetherIncrease in insulin resistance environment；As Reason-study, test CaMKII γ shortage pair (b) Advanced lesionsThe influence and mechanism of Apoptosis and patch necrosis.Use the overall plan identical with previously described (76) It omits, 4 groups of mouse will act as carrying out the donor of bone-marrow transplantation (BMT) into male Ldlr-/- mouse below：Camk2gfl/ flInsr+/+(WT→Ldlr-/-)；Camk2gfl/flLysmcre+/-Insr+/+(-CK KO→Ldlr-/-)； Camk2gfl/flInsr-/-(-InsR KO→Ldlr-/-)；With Camk2gfl/flLysmcre+/- Insr-/- (- CK/InsR DKO→Ldlr-/-).All genotype have all been returned in C57BL6/J backgrounds>8 generations, and all experiments will all make Use littermate control.Previous research have shown neuropathic in Lysmcre+/- backgroundIn floxed genes almost lack It loses (67,97).6 weeks after BMT, for mouse by feeding Western diet (WD) 8,12,16 and 20 weeks, it is early that this will make it possible to analysis Phase, interstage and late period/necrotic lesion (16,65,67,71,97-102).The first prerolandic artery Rolando carried out with similar strategy and target Atherosis research (16,65,71,97,101,102) allows to estimate together with the statistic from previous In vivo study (76) Our 80% chances will be given to detect 33% difference of these terminals between four groups of mouse by counting every group of n=30 male mice.
Be described in detail in publication in this field for the research of rat aorta atherosis (includingApoptosis and Necrotic cores quantify) overall plan (16,65,67,71,72,97,101-103).In brief, blood plasma is obtained to carry out fat Matter/lipoprotein analysis, fasting glucose and insulin analysis.Analyze the lesion and necrosis area of aortic root and brachiocephalic artery； Apoptosis (TUNEL and the caspase-3 mRNA of activation)；It is buried with born of the same parents in situ.The born of the same parents in situ measured in lesion are buried (103).It is bad Unleavened dough product is defined as havingFragment is (in the case of there is no cellSpecific antigen) acellular area and It is obtained in the case of being dyed there is no collagen., SMC, EC and T cell will identify that and use is directed to by IHC The valve Huo Fushi of elastin laminin (elastin) and collagen (Verhoeff ' s) and horse Xun Shi (Masson ' s) trichrome stain The thickness (97) of agent basis weight of fiber lid.Aftermentioned measure will be with such as described (104) by detecting 20 weeks lesions with degradable valve Inner elastic fiber (" inside erosion ") in the sample of Hough dyeing is supplemented, because containmentSecondary necrosis can be led The reason for cause less leakages of MMP, this can be this terminal (105).Such as (104), data show lesion in quantification of each group Show the number for the mouse that inside is corroded.Positive findings will be followed by such as (106), using MMP gelatinases (MMP2/9) substrate, The active MMP that lesion is cut into slices is measured by enzyme spectrum analysis and near-infrared fluorescent.Pi Ershi (Perl's) NiHCF thin films agent will be used (109) and the H＆E cut sections for microscopic examination for RBC and 560nm fluorescence (107,110) measure the advanced lesions of patch internal haemorrhage Matter (107,108).Using anti-F4/80 () and the IHC of anti-p-CaMKII whether will be used to assess p-CaMKII with patch rank Section promote and increase and whether in deficiencyIncrease in insulin signal transduction environment.From Camk2g-/- mouse 'sIn, anti-p-CaMKII antibody does not give the signal higher than background.As people's lesion (Figure 17), late period disease has been obtained DenaturationIn with that there are the consistent dyeing patterns of p-CaMKII, and will use- CK KO groups verify this signal.It will be fixed Measure p-CaMKII stained area of the IHC data as each lesion area；AsCaMKII γ staining cells percentage Than；With p-CaMKII dyeingPercentage.It will be to exempting from from the aortic root and arched roof section of its removal outer membrane Epidemic disease trace is so that can precise Identification p-CaMKII (and total CaMKII).This analyzes the influence for being not excluded for other arterial wall cells, But whether IHC researchs should allow us to assess the γ hypotypes that these other cells even express the enzyme.It will carry out pro-inflammatory (TNF α and IL-6) and anti-inflammatory (TGF β and IL-10) mRNA expression of cytokines laser traps microdissection (LCM)-RT- QPCR, becauseApoptosis and ER stress reduction will likely reduce plaque inflammation (70,111), and there is no CaMKII γ can reduce the transcription of inflammatory gene (53,54) by the way that NCoR is made to be total to repressor function-stable.The RNA of trapping will be also used for Probe into the internal mechanism as instructed by cell culture studies.The rush Apoptosis that the CaMKII γ of activation will be assessed transcribes target Mark, particularly Fas, Nox2 and Chop (Ddit3) mRNA48.Also Serca2b, Ikbe and Bcl2 will be measured (in insulin resistanceThe NF- κ B targets 66 of middle reduction) mRNA.It will measure as FoxO1 targets (112)The mRNA of Tlr4 and Tlr2 Expression.On Nox2 and oxidative stress (93), neuropathic ROS will be measured using DHE.These results will show (a) compared with WT Group, the advanced lesions in CKO groupsApoptosis and the label of patch necrosis and CaMKII- apoptotic pathways will It is relatively low, and especially with respect to the CK/InsRDKO groups of WT groups；(b) compared with WT groups, in CKO groupsFas, Nox2 and Chop mRNA and ROS will be relatively low, and especially with respect to the CK/InsR DKO groups of WT groups；And (c) compared with WT groups, pancreas Relatively low and Ikbe higher will be caused and be concerned with observing these trend in CK/ by the Serca2b and Bcl2 in the element resistance group of island Whether reversed in InsR DKO groups.
Table 1：The general introduction of mouse model and end-point prediction
Compared with WT groups, the label of necrosis and CaMKII- apoptotic pathways will be relatively low in CKO groups, and especially It is the CK/InsR DKO groups compared with WT groups；(b) compared with WT groups, in CKO groupsFas, Nox2 and Chop mRNA with And ROS will be relatively low, and especially with respect to the CK/InsR DKO groups of WT groups；And (c) compared with WT groups, insulin resistance group In Serca2b and Bcl2 by relatively low and Ikbe higher, and will cause and be concerned with observing these trend in CK/InsR DKO groups In whether reverse.
Characterization liver CaMKII γ lack mechanism and the measure liver specificity for improving that the metabolic disorder of obesity is relied on CaMKII γ lack the atherosclerosis that whether will contain obesity mice.
HC CaMKII γ-in ER stress-inducedsThe same enzyme to play a crucial role in Apoptosis-shortage change Atherogenic metabolic disorder in kind obesity.CaMKII γ are the main CaMKII hypotypes in mouse and people liver and compare In thin control, the p-CaMKII γ of the measurement as the CaMKII γ states of activation are small in (DIO) that ob/ob mouse, meals induce Mouse (5.24 kilocalories/g, 60% calorie from fat, x20 week) and the liver of morbid obesity people in increase (Fig. 8 A-B).With these Data and obesity cause liver ER stress (34) the fact it is consistent, the ER reached by azetidine or palmitate stress activate increasing Add the p-CaMKII γ in HC, and the CaMKII γ in DIO mouse lack containment UPR activation, so as to show to exist as CaMKII γAs middle performance, it participates in the UPR positive feedback loops in HC (referring to Figure 16).It is small compared to DIO WT by finding Mouse, although weight indifference, the DIO mouse that CaMKII γ lack have significantly lower plasma insulin and to insulin-mediated The response bigger that reduces of glucose show functional importance (Fig. 9 A-D) of the CaMKII γ in obesity.In grape During sugared tolerance is tested, the blood glucose of mouse also has improvement.By using the gland-K43A- for being verified inhibition liver CaMKII CaMKII carries out acute inhibition to liver CaMKII and obtains similar results (Fig. 9 E-F), and the gland-K43A-CaMKII is that kinases is not lived Change the CaMKII123 of dominant negative form.In the liver of the ob/ob mouse of gland-K43A-CaMKII processing, gluconeogenesis (GNG) base Because the mRNA of G6Pase and PEPCK reduces (Fig. 9 G).
As the Igfbp1 reduced in the liver also inhibited in CaMKII, two kinds of genes are the targets of FoxO1.These Data and the CaMKII of containment FoxO1 nuclear locations inhibit consistent (see below).The DIO mouse that CaMKII γ lack also have drop Low (a) liver triglycerides (TG) content (Figure 10 A)；(b) SERBP-1c induction fat generation mRNA rather than with aliphatic acid oxygen Change relevant mRNA；Plasma cholesterol and TG (Figure 10 B) (c).The dyslipidemia of high fat diet induction is related to by acting on The hyperinsulinemia of remaining actrapid monotard's receptor and stimulate mTORC1-pS6K approach, so as to cause containment sorting protein 1 (Sort).Sort reduce with people's dyslipidemia genetic association, and mechanism seemingly Sort mediation LDL apoB-100 degradation It reduces, the degradation is perhaps the lysosomal degradation pathway 124 mediated by Sort chaperones.This approach passes through CaMKII Inhibit and be partly obstructed (Figure 10 C).Finally, compared with WTDIO livers, the liver Tnfa mRNA in Camkg2g-/- liver are reduced about 50% (Figure 10 D).
Liver cell CaMKII γ lack reduces gluconeogenesis (GNG) and hepatic glucose by being related to the mechanism that FoxO1 is arranged outside core (HGP) is generated to mitigate hyperinsulinemia and secondary dyslipidemia(Figure 18).
This analysis raises effect and hyperinsulinism in the hyperinsulinemia in promoting obesity based on (a) HGP Importance of the mass formed by blood stasis in dyslipidemia is driven；(b) CaMKII in obesity mice inhibits to reduce the GNG genes of FoxO1 inductions With the result of study (Fig. 8 G) of FoxO1 targets Igfbp1；(c) on liver CaMKII γ, the FoxO1 in fasting slight of stature mouse and Associated data between GNG.
It will be lacked at present with alpha1-antitrypsin Cre (A1atcre+/-) mouse hybrid in HC using as described herein CaMKII γ Camk2g mouse 125.Four groups of male mices will start feeding DIO meals 20 weeks in 4 week old：DIO/ Camk2gfl/fl (DIO/WT) and standard diet feeding control (thin/WT)；With DIO/Camk2gfl/flA1atcre+/- (DIO/ Li-CK KO)；(thin/Li-CK KO) is compareed with standard diet feeding.To exclude the compensating effect that system genitale knocks out, one group of DIO/ Li-CK KO mouse will use gland-T287D-CaMKII (to compare) " recovery " compared with gland-LacZ, the gland-T287D-CaMKII It is the constitutive activity form (123) of the kinases, the prediction beneficial metabolic in DIO/Li-CK KO mouse should be reversed to act on. Food intake and weight will be monitored, and fasting plasma glucoses and insulin were obtained by every 4 weeks.It is small at the end of feeding period Mouse will be fasted 6 it is small when, weigh and put to death.In view of circadian rhythm with metabolism associate (126-128), will strictly comply with 12 it is small when Illumination/dark cycle, and all mouse will all be condemned to death in the same time of one day.Although Camk2g-/- mouse can be without bright Aobvious morphology or function sexual abnormality (including with the relevant exception of liver) in the case of be developed to the manhood 48, but will obtain blood plasma with It measures following metabolism and lipid parameter and the alanine aminotransferase (ALT) and aspartic acid of the measurement as HC dysfunctions turns Ammonia enzyme (AST).Liver will then remove at once, and snap frozen is stored in liquid nitrogen under -80oC.The missing of CaMKII γ will Confirmed by RT-PCR and Western blotting, and the snap frozen sample to brain, heart, kidney, adipose tissue, skeletal muscle and intestines will be passed through This progress similar analysis come measure liver missing specificity.Also p-CaMKII, p-JNK134 of immunoblotting assay liver will be passed through With UPR labels (34), and in the case of hepatitis disease, pass through the Tnfa that QPCR analyzes liver.Under without being bound by theory, relatively These terminals in thin WT, DIO/WT groups will raise, but be contained in DIO/Li-CKKO groups.
HC CaMKII γ, which lack, improves glucose and lipid-metabolism
8 mouse in each group will be subjected to the research of complete hyperinsulinemia-euglycemic clamp, and the research will be into One step assesses the basic and insulin stimulating in insulin sensitivity and quantitative peripheral tissues (for example, skeletal muscle, adipose tissue) Hepatic glucose generate glucose uptake, glycolysis and the Glycogen synthesis (129) of (HGP) and insulin stimulating.Compared with thin WT groups, DIO/WT groups should show that glucose infusion reduces, HGP increases and peripheral tissues' glucose disposal reduces, so as to reflect this Insulin resistance in a little mouse.Compared with DIO/WT mouse, these parameters in DIO/Li-CK KO mouse will improve (i.e., Glucose infusion increases and HGP is reduced).Every group of 8 mouse can show DIO/Li-CK KO groups and DIO/ with 80% effect Statistically significant difference (P between WT groups<0.05).
Compared to DIO/WT mouse, DIO/Li-CK KO mouse will have the distribution of smaller atherogenic lipid, described Distribution will be surveyed by measuring plasma TG, T-CHOL, HDL- cholesterol, the distribution of FPLC lipoprotein and Atherogenic Examination and the rise (130) under insulin resistant state.For supplement blood plasma research, the TG for analyzing the liver sample from mouse is contained Amount, ldl receptor protein matter and mRNA and the Srebp1c and its transcriptional targets for analyzing mRNA.Compared with DIO/ mouse, The prediction reduction of the plasma lipoprotein rich in TG in DIO/Li-CK KO mouse is attributable to liver secretion reduction or liver removes and increases Add.To measure TG secretions, triton WR-1339 researchs (131) will be carried out.In brief, the mouse of fasting will use [35S]-first sulphur Propylhomoserin is injected to inject that lipoprotein is blocked to remove by metabolic marker apoB and with triton WR-1339.It will be 2 after entire injection Blood plasma is collected during period hour and analyzes the TG contents and VLDL of blood plasma, and will pass sequentially through SDS-PAGE and autoradiograph To measure apoB100 and apoB48.Increase rate of the lipoprotein of the apoB marks of mark when 2 is small during period is secretion speed The measurement of rate.CaMKII γ shortages, which express the liver Sort in insulin resistance environment, increases (derepressing) (Figure 10 C).Target exists It is secreted in determining to make the liver Sort silences in DIO/Li-CK KO mouse whether to increase VLDL.The fatty liver of DIO mouse because CaMKII γ lack and are significantly improved (Figure 10 A).By the oil red O in the liver sample for measuring 4 groups of mouse of TG contents and dyeing Positive lipid droplet.If the display liver TG accumulation of DIO/Li-CK KO mouse reduces, then gene will be generated by measuring fat, Including Srebf1, Fasn, Acaca, Scd1；Gene involved in fatty acid oxidation, including Ppara and Acox1；And in view of it The expression of the Nampt of the effect in steatosis in FoxO KO Mouse Livers detects mechanism (132).Follow-up study will be visited Studying carefully makes CaMKII and the associated molecular mechanism of positive result of study, as the result by testing above is instructed.For example, preliminary data It supports associating between UPR effectors ATF3 and mTORC1-SORT ways to curb crime, and therefore gives CaMKII γ amplifications UPR's Ability.
Obesity is promoted by CaMKII γ phosphorylations
In, ER stress by be related to CHOP induction ER Ca2+ storage caverns discharge pathway activation CaMKII γ (48, 68).In view of in obesity, liver ER stress be fatty in the liver of ER stress-induceds by induction (34) (above) and CHOP with CHOP The reason in denaturation property acts on (94), whether required by the CaMKII γ activation in obesity will test CHOP.Prepared with Pure C57BL6/J is the Chopfl/fl mouse of background.When mouse and foreign gene remove (deletor)-Cre mouse hybrids, Generation CHOP is lacked.These mouse will be with A1atcre+/- mouse hybrid to test compared to compareing Chopfl/fl mouse, these Whether mouse has less liver p-CaMKII γ under DIO meals (referring to Fig. 8).If it were to be so, it and if is using It is above measure under, glucose and lipid-metabolism in these mouse are improved, then they will use constitutive activity gland- T287D-CaMKII transduces to show that the beneficial effect that liver CHOP lacks is reversed.Tracking metabolism and mechanism experiment will be with laggard Row, it is as a result undetermined.
GNG is promoted by CaMKII.
Fasting promote the phosphorylation of CaMKII γ and this reduced (Figure 19 A) by feeding again.It is consistent with these data, pancreas Glucagons (and forskolin) promotes the CaMKII γ phosphorylations in primary HC.By fasting Camk2g-/- mouse or with gland- The blood glucose that K43A-CaMKII processing is stimulated with the fasting in the WT mouse of more acute inhibition liver CaMKII and pyruvic acid reduce and Liver G6pc and Pck1mRNA reduce to show in vivo functionality importance (Figure 19 B-D).In fasting liver or serum starvation HC CaMKII γ lack or inhibit that core FoxO1 is also caused to significantly reduce (Figure 19 E-F), and constitutive activity T287D CaMKII make core FoxO1 (Figure 19 F), plasma glucose and insulin and liver G6pc and Pck1mRNA increase.CaMKII γ lack to G6pc The containment of mRNA, which is acted through, is transduceed with composing type core FoxO1-ADA and is able to abolish (Figure 19 G), this work lacked with CaMKII With needing, the idea that FoxO1 is arranged outside core is consistent.It is anti-in view of the FoxO1 phosphorylations at T24, S316 and S253 of Akt inductions Its road and go, it is that kinases can promote core FoxO1 (133) that can seem inconsistent.In fact, CaMKII γ shortages do not promote at this Phosphorylation at a little sites.However, LC-MS phosphoeptides Position Research (134) show FoxO1 in two other sites- The phosphorylation of Ser284 and 295-in the serum starvation HC from WT mouse but from Camk2g-/HC of-mouse in completely Non- phosphorylation (Figure 19 H).Therefore, there is no pSer284/295 under without being bound by theory, in the HC that CaMKII lacks to promote It is arranged outside FoxO1 cores and thus contains GNG.
The G6pc and Pck1mRNA and core FoxO1 that will be measured in 4 groups of mouse.Compared with DIO/WT mouse, the two exists To it be contained in DIO/Li-CK KO mouse.FoxO1 from mouse will be subjected to immune precipitation to carry out phosphoeptide positioning. CaMKII shortages will be associated with there is no pSer284/295-FoxO1.S284/295A-FoxO1 can simulate CaMKII γ inhibition Effect (↓ core FoxO1 and G6pc and Pck1mRNA), and the effect that S284/295DFoxO1 replies CaMKII γ inhibit has Resistance.The HC of the liver specificity FoxO1KO mouse of GNG125 with reduction builds the WT marked with GFP or mutation FoxO1 Body ± gland-K43A-CaMKII transductions will then measure FoxO1 positioning and G6pc and Pck1mRNA to inhibit CaMKII.For To use K43A-CaMKII inhibit CaMKII supplement, the HC from Li-CK KO will use gland-WT-FoxO1 compared with gland- S284/295D-FoxO1 transduces.Under without being bound by theory, this mutant will be constitutively in core and to CaMKII γ The ability for lacking containment GNG has dominant negative effect.For general introduction：
Table 2
Ad., adenovirus；Control, ad-LacZ；K43A, dominant negative CaMKII；S → A FoxO1, composing type dephosphorization garden sorrel Type；S → D FoxO1, composing type " phosphoric acid " model
These predictions will transfer to the L-FoxO1 mouse for the DIO feedings that more than adenovirus construct is used to transduce.Terminal will be Liver G6pc and Pck1mRNA；Core FoxO1 when applicable；Plasma glucose (after being excited including pyruvic acid) and insulin；On and State lipid and lipoprotein parameter.Prediction is similar to those in table, wherein the reduction of GNG genes will with glucose and lipid/ The improvement of lipoprotein metabolism.In the case of impacted but G6pc and Pck1 is impregnable unlikely in HGP, GNG will be measured Gene Pdk4, Fbp1 and Gck.
The activation of CaMKII γ in the liver of obese people.
It will check from the obese subjects for being subjected to bariatric surgery and from the thin subject for being subjected to generally performing the operation The fresh liver biopsy samples of snap frozen.There will be no patient's identifier, but there will be the coding inventory for each subject, Including weight, waist:Stern ratio, age and gender；Diabetes/metabolism drug；Fasting plasma glucose, insulin, HbA1c and free Aliphatic acid；And based on histologic analysis, the degree of steatosis and steatohepatitis (according to scale from 1 to 5 classification).To examination Sample carries out Western blotting to measure p-CaMKIIγWith total CaMKII γ and ER stress label, including CHOP and Sort.Data It will compare compared with loading, be quantified by densitometry analysis.Main target is to determine compared with thin subject, to fatten oneself Whether the p-CaMKII in the liver of fat subject is higher and deteriorates in fat group with the presence or absence of p-CaMKII with insulin resistance And increase.By-end is to determine whether high-caliber p-CaMKII is related to high-caliber CHOP and low-level Sort Connection.The p-CaMKII of each thin liver sample of result (Fig. 8 B) display as described herein is horizontal less than each fat sample, and therefore, Plan sample size for fat liver and the n=20 of thin liver should have>95% effect shows thin source property sample and fat source There are statistically significant difference (P in terms of the level of p-CaMKII between property sample<0.05).For p-CaMKII in fat liver with Associating between insulin resistance and between p-CaMKII and CHOP and Sort expression, 20 fat liver samples will provide 80% Effect come find related coefficient 0.6 have statistical significance.Further research will by Mechanism Study (for example, in previous section FoxO it is relevant those) guidance.
Liver In insulin resistances and glucose and lipid-metabolism of the CaMKII γ in obesity environment in rise Effect.
HC expression in obesity mice attracts the Chemokines CC CL2 (MCP-1) of new bone marrow cell, the bone marrow cell with Afterwards by promoting aliphatic acid esterification and the genetic transcription involved in TG accumulation play an important role in fatty degeneration of liver (45).This Outside, the liver of the activation including Kupffer cellCan HC fatty acid oxidations (44) be contained by paracrine mechanism.Although HC CaMKII γ can help to attract new, but under without being bound by theory,CaMKIIγIt can beMediation to HC lipids It plays an important role in the effect of metabolism.Specifically,The saturated fat acid activation that can be exposed in fat liverIn UPR and UPR activation withInflammation is associated (72,111).If it were to be so, it and is being mediated in view of CaMKII γ And expansionIn ER stress during effect (Figure 16), liverCaMKII γ can be to the HC lipids in obesity environment Metabolism and insulin resistance have material impact.As described herein, in the suppressed fat livers of CaMKII, TNF α reduces (figure 10D), and CaMKII γ lack withInflammation mitigates associated (53,54).It will carry out similar DIO to study, wherein will substitute A1at-Cre mouse use LysMCre mouse.IfCaMKII γ lack the metabolizing parameters improved in obesity environment, bag Include steatosis, then by checking mechanism.Main concept is ER stress-inducedsInflammation can be relatively light, and therefore, will pass through LCM-RT-QPCR measures liverUPR and cytokines mRNA.Theoretically, if such as CaMKII γ shortages make monocyte Chemokine receptor expression reduces, then liver also may be presentNumber reduce.Therefore, will useIHC quantifies liver Content.Further Mechanism Study will be by data-driven.For example, evidence suggests PPAR δ activation can be in obesity environment LiverIn a kind of improvement process (44), will evoke research the liver from separate groups of miceIn CaMKII, ER should Swash, the associated experiment between inflammation and PPAR δ approach.
With the relevant atherosclerosis studies of liver CaMKII γ.
Liver CaMKII γ lack by improving dyslipidemia and possible other parameters (for example, inflammation, cycle FFA, blood pancreas islet The excessive arterial wall effect of element and hyperglycaemia), the atherosclerosis in insulin resistance environment will be mitigated.Western diet (WD) Feeding Ldlr-/- mouse is needed for atherogenic lipoprotein phenotype.In C57BL/6 backgrounds, feeding fat content is only suitable Degree causes insulin resistance 135 and the phosphorylation of liver CaMKII γ as shown in Figure 20 less than the WD of DIO meals.To assess this A model will make MOUSE REPRODUCTION to obtain following 2 groups：Camk2gfl/flLdlr-/- (LDLR KO) and Camk2gfl/ flA1atcre+/-Ldlr-/-(Li-CK LDLR DKO).With WD8, after 12,16 and 20 weeks, metabolizing parameters, blood will be measured Slurry and inflammatory cytokine and FFA.It was to be enough the time (135) for developing insulin resistance in this model with WD8 weeks. Will focus on can be by lesion terminal that these factors are aggravated.It is originated for lesion, endothelial activation will be measured (for example, acetylcholine Dependence vasodilation, VCAM-1 expression, the adhesion of p-eNOS, ROS and monocyte to endothelium) andAccumulation.These are With dyslipidemia (136), hyperglycaemia (137) and increased FFA and insulin resistance to the effect of endothelium (130,138, 139) associated process.For plaque progression, plaque cells will be measured (especially) in expression of pro-inflammatory cytokine mRNA 28；It can be triggered by saturation FA and insulin resistanceApoptosis (72,73)；It can be deteriorated in obesity by saturation FA Deficiency born of the same parents bury (140)；And the patch internal haemorrhage (141) in advanced lesions associated with the dyslipidemia of diabetes.
LiverIn can improve insulin sensitivity and glucose and lipid-metabolism there is no CaMKII γ.In that feelings Under condition, LysMCre models can have that there are two types of for improving the mechanism of atherosclerosis.Therefore, will testWith HC Whether lacking for both CaMKII γ will have notable protective effect to the atherosclerosis in all stages.For this mesh , by additional 2 groups be added to research as described herein in, wherein Li-CK LDLR DKO mouse use come from compared with The bone-marrow transplantation of the WT mouse of Camk2gfl/flLysmcre+/- mouse.This will be by testing atherosclerosis and metabolism All aspects are assessed.
Table 3
The general introduction of mouse model and end-point prediction：
On KO models, control will be added by including gland-T287D-CaMKII → DIO/Li-CK KO groups.If The phosphorylation of FoxO1Ser284/295 is not related in the metabolism of mediation CaMKII, then parameter will be probed into, including influencing FoxO1 positioning involved in core input/output means those, such as with the interaction in 14-3-3 sites (142)；And with CaMKII γ it is relevant to the effect of the possibility of liver insulin receptor signal transduction path those.If Ser284/295 phosphorylation weights It will, then future studies will inquire into whether (a) CaMKII γ are directly related to or whether it induces another kinases to reach this mesh (143)；And how the defects of (b) these sites type phosphorylation can promote core to export FoxO1.FoxO1-S284/ will be generated 293A mouse are to study these effects of p-Ser residues in hepatic glucose and lipid-metabolism.Mitigate CaMKII γ and lack environment In ER stress can improve responses of the HC to acute insulin.For example, containment ER stress can reduce the Ser207 phosphorylations of IRS1, This improves insulin signal transduction (34).
Embodiment 8：Hepatic glucose when being in fasting and obesity by the CaMKII Ca2+ oscillations transduction regulatories carried out produces It is raw
Hepatic glucose generation (HGP) is most important for glucose homeostasis, but potential mechanism is not yet explained completely It is bright.It is shown here be calcium sensitivity enzyme CaMKII in primary HC by cAMP and glucagon and in vivo by pancreas hyperglycaemia Element and fasting are activated in a manner of calcium and IP3R dependences.The heredity of CaMKII lacks or inhibits the phosphorylation by influencing FoxO1 To block the decomposition of glycogen and gluconeogenesis of the core transposition of FoxO1, damage fasting and glucagon/cAMP inductions, and reduction blood Sugar level, and constitutive activity CaMKII has adverse effect.It is important that the containment that CaMKII lacks to glucose metabolism is made It is abolished with being transduceed by using composing type core FoxO1, so as to indicate that the effect that CaMKII lacks needs to arrange outside core FoxO1.This identical approach is directed in the excessive HGP in obesity environment.These results disclose FoxO1 nuclear locations and liver The signal transduction path that calcium involved in glucose homeostasis mediates.
Main points include：
Fasting and glucagon activate liver Ca Μ Κ Ι Ι in a manner of PKA-IP3R1-Ca2+i dependences
CaMKII promotes the hepatic glucose of fasting/glucagon induction to generate (HGP)
CaMKII promotes the HGP for CaMKII mediations to stimulate required core FoxO1.
CaMKII-FoxO1 approach is directed in the excessive HGP in obesity environment.
Liver is responsible for maintaining orthoglycemic major organs under the conditions of nutritional losses.During the early stage of fasting, Liver transfers glucose (Radziuk and Pye, 2001) using glycogen storage cavern.As fasting is in progress, glucose is from non-carbon hydrate The recombining of object precursor (gluconeogenesis) become hepatic glucose generate (HGP) it is main facilitate process (Lin and Accili, 2011).These variations are conducted in response to direct hormone signal and quickly occurred.In addition, insulin and glucagon are both It influences the G-6-Pase (G6pc) involved in both gluconeogenesis and decomposition of glycogen and also regulates and controls the phosphoenol third of HGP Ketone acid carboxylic kinases (Pck1) transcription (Pilkis and Granner, 1992；Burgess etc., 2007).During fasting, such as FoxO The variation of the Subcellular Localization of " glucose generative nature " transcription factor of (1,3 and 4) and Crct2 activates the expression of these genes (Lin and Accili, 2011).In addition, receptor-gamma co-activating -1 α of son (PGC-1 α) such as peroxisome proliferation activation With CBP's different co-activating be considered with cAMP response component (including CREB, 4 α of hepatocyte nuclear factor (HNF4 α), Sirt1 and Clock gene) interaction, so as to cause gluconeogenesis gene transcription increase (Hall etc., 1995；Matsumoto etc., 2007； Puigserver etc., 2003；Rhee etc., 2003).Except excessive glucagon signal transduction is stimulated during fasting in HGP Outside effect, it be recognized as playing an important role in the hyperglycaemia in diabetes B (Sorensen etc., 2006；Unger and Cherrington,2012；Saltiel,2001).
Glucagon stimulates in general HGP transcription factors, and specifically stimulating the core of the FoxO1 of HGP easy Position by intracellular signal transduction approach be not fully understood.In this scenario, to making intracellular Ca2+ (Ca2+ i) and sugar The regulation and control of heteroplasia are associated be previously reported it is interested (Friedmann and Rasmussen, 1970；Kraus-Friedmann and Feng,1996；Marques-da-Silva etc., 1997).For example, glucagon and cAMP can increase Ca2+ i, and shown Ca2+ iChelating can reduce glucagon induction HGP gene expressions and glucose generate (Bygrave and Benedetti, 1993；Staddon and Hansford, 1989；Mine etc., 1993).Not there is provided based on these makes Ca2+ iPhase is metabolized with hepatic glucose The previous research of associated molecular mechanism under without being bound by theory, imply Ca2+ iSensitivity enzyme CaMKII has effect.
Calcium Calmodulin depedent kinase II (CaMKII) is a kind of Ca as in cell2+ iImportant Jie of signal transduction Lead object serine-threonine kinase (Couchonnal and Anderson, 2008；Singer,2011).There are four kinds of CaMKII Hypotype-α, β, the γ and δ-independent gene code of each freedom.α and β hypotypes are mainly in neuron, and CaMKII γ and CaMKII δ is expressed in extensive Various Tissues.After calcium/calmodulin complex is combined, autophosphorylation on Thr287 generate calcium/ Calmodulin inde-pendent activity (Couchonnal and Anderson, 2008；Singer,2011).On the most of CaMKII Number research carries out in neuron and cardiac muscle cell, and only exists limited understanding to the CaMKII in other tissues, wherein so far Until the present not with the relevant understanding of glucose metabolism.In current research, display CaMKII activity is by cAMP and pancreas hyperglycaemia Element increases and also responds to fasting in vivo and increase.What is further illustrated is regulation and control of the CaMKII in decomposition of glycogen and gluconeogenesis In play a major role.Specifically, following evidence is provided：CaMKII in vitro and in vivo with two kinds of key enzyme G6pc of regulation and control and The mode of the expression of Pck1 has profound influence to FoxO1 nuclear locations.Finally, present and indicate that this identical approach is related in obesity The evidence in excessive HGP in disease environment.
As a result
Glucagon and fasting are with IP3R and Ca2+ iDependence mode activates liver CaMKII
Show that glucagon increases the intracellular Ca2+ (Ca in liver cell (HC)2+ i) (Staddon and Hansford, 1989), this be able to recently verification (Y.Wang, G.Li, J.Goode, J.C.Paz, R.Screaton, W.H.Fischer, I.Tabas and M.Montminy, manuscript are submitted for delivering).To determine whether glucagon activates Ca2+ iSensitivity enzyme Primary mouse HC is handled various periods and then measure CaMKII enzymatic activitys and at Thr287 by CaMKII with glucagon CaMKII phosphorylations, the phosphorylation be the CaMKII states of activation measurement (Couchonnal and Anderson, 2008； Singer,2011).Two kinds measure results show CaMKII activity increase with glucagon processing time (Figure 1A- B).Double [2- amino-benzene oxygens] ethane-N, N, N ' of cytosol calcium chelating agent 1,2-, N '-tetraacethyl four [acetoxymethyl] (BAPTA-AM) it is used to determine Ca2+ iEffect and discovery BAPTA-AM to CaMKII activation significantly reduce glucagon induction CaMKII phosphorylations (Fig. 1 C).
1,4,5- inositol triphosphate receptors (IP in endoplasmic reticulum (ER)3R) channel response is in IP3With reference to and discharge Ca2 +, and Ca in the cell2+ iIt plays a major role in homeostasis.Other researchs have revealed that the PKA of glucagon induction makes IP3R Phosphorylation and increase IP3R activity, so as to cause Ca2+ iIncrease (Y.Wang, G.Li, J.Goode, J.C.Paz, R.Screaton, W.H.Fischer, I.Tabas and M.Montminy, manuscript are submitted for delivering).Also shown that glucagon can inducing phospho The IP3 releases (Hansen etc., 1998) of enzyme C mediations.Work in the CaMKII activation induced for research IP3R in glucagon With using the smooth Spongistatin C processing of IP3R inhibitor from Ip3r1fl/flThe HC of mouse and as compensation process, gland-Cre Processing comes from Ip3r1fl/flThe HC of mouse.The IP3R1 missings of smooth Spongistatin C processing and Cre mediations both cause pancreas height The CaMKII phosphorylations of blood glucose element induction significantly reduce, so as to illustrate that IP3R has key effect (Fig. 1 D) in this process.
Glucagon receptor signal transduction, including Ca2+ iInvolved in increase signal transduction (Staddon and Hansford, 1989), it is to generate cAMP, with post activation as the key enzyme involved in HGP by adenosine cyclase of acid Protein kinase A (PKA) mediate.In this scenario, the work with the bromo- cAMP processing HC simulation glucagons of 8- is found With and phosphoric acid CaMKII is caused to dramatically increase (Fig. 1 E).In addition, work as before glucagon is added, at PKA inhibitor H89 When managing HC, the phosphoric acid CaMKII increases of glucagon mediation are significantly inhibited (Fig. 1 F).These data support that there are a kind of ways The intracellular Ca that footpath, wherein glucagon-cAMP-PKA signal transductions mediate IP3R by it+2The effect of release promotes Phosphorylation/activation of CaMKII.
To check whether CaMKII is regulated and controled in vivo by glucagon, (i. in mouse peritonaeump) glucagon Bolus excites.Consistent with the effect observed in the HC of culture, liver CaMKII phosphorylations are handled by glucagon and induced (Fig. 1 G).It was found that down to 1 μ g kg-1Glucagon dosage can make CaMKII phosphorylations (Figure 28 A) in liver.To obtain It is important internal evidence in the regulation and control for the CaMKII phosphorylations that IP3R is mediated in glucagon, the smooth Spongistatin C of mouse Processing 4 days in peritonaeum.Then mouse is excited with glucagon, and measures the p-CaMKII of liver extract.As shown in figure iH, Smooth Spongistatin C processing significantly reduces the CaMKII phosphorylations of glucagon induction.Then, compare from feeding to fasting shape The liver CaMKII phosphorylations of the transition period of state, it is known that the period can make Plasma glucagon rise (Lin and Accili, 2011) (Figure 28 B).After data are shown in fasting, liver CaMKII phosphorylations dramatically increase, and the total amount of CaMKII seems from battalion The state of supporting influences (Fig. 1 I).In addition, after feeding again, the horizontal of p-CaMKII in liver reduces (Fig. 1 J).Such as pancreas hyperglycaemia Element processing is the same, and the phosphorylation of the CaMKII of fasting induction is by smooth Spongistatin C processing mouse and by containment (Figure 28 C).These The activity of data display liver CaMKII tune in a manner that the latent effect in the HGP with being induced in fasting is consistent by nutritional status Control.
CaMKII promotes the glucose in primary HC to generate
CaMKII γ are the main CaMKII hypotypes in HC, and in the HC for lacking γ hypotypes, other hypotypes are not induced (Figure 22 A).In view of liver CaMKII activity is regulated and controled in vivo by glucagon and fasting, measure from WT mouse and Camk2g-/- Glucose in the HC of mouse generates.Cell, the forskolin are checked under primary condition and after being stimulated with forskolin It is glucagon analogies and strength adenyl cyclase activator (Harano etc., 1985).Data are shown in CaMKII γ In the HC of shortage, the glucose of primary condition induction, which is generated, to be generated both with the glucose of forskolin induction by containment (figure 22B).Then the adenovirus (gland-CA-CaMKII) with expression constitutive activity CaMKII, expression " kinase-dead are checked (kinase-dead) " adenovirus (gland-KD-CaMKII) (Pfleiderer etc., 2004) of dominant negative CaMKII or expression Glucose in the WT HC of the adenoviral transduction of LacZ controls generates.CA-CaMKII has 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor T287D, simulation Autophosphorylation at T287 and the autonomous activity of generation under calcium/calmodulin there is no combination, and KD-CaMKII is swashing There is disabling mutation (Pfleiderer etc., 2004) in enzyme domains.It observes in the cell with gland-CA-CaMKII transductions Primary condition induction glucose release with forskolin induction glucose release both increase (Figure 22 C and 28D).With The HC of gland-KD-CaMKII transductions, which shows that the glucose of forskolin induction generates, reduces (Figure 22 C), described to cause CaMKII Activity reduces about 40% (Figure 28 E).
CaMKII evokes the effect of HGP us and studies to the enzyme of encoding regulator HGP, i.e. G-6-Pase and phosphorus The transcription of two kinds of genes of sour enolpyruvate carboxylic kinases influences.For this purpose, measure G6pc the and Pck1mRNA water in above-mentioned model Flat (Figure 22 D-E).In all cases, knock out or the CaMKII of KD-CaMKII mediations inhibits to reduce forskolin or the high blood of pancreas The gene expression of sugared element induction, and CA-CaMKII increases gene expression.Under there is no forskolin or glucagon, WT HC In G6pc and Pck1mRNA expression it is more much lower than the expression in the WT HC of HORMONE TREATMENT, but even at these Under the conditions of, CaMKII γ shortages also cause gene expression to reduce (Figure 28 F).In addition, gland-KD-CaMKII does not reduce shortage Relatively low but detectable horizontal (Figure 28 G) of the G6pc mRNA of forskolin induction in the HC of CaMKII γ, this in Figure 22 E KD-CaMKII is since the premise of CaMKII inhibition is consistent to the containment effect of G6pc.
In short, CaMKII, which lacks and inhibit data, shows that endogenous CaMKII is generated and Pck1/G6pc genes in glucose Importance in expression, and the data on CA-CaMKII are shown when enzyme is expressed at a high level, it can be there is no hormones In the case of promote these processes or enhance these processes in the presence of hormone.
Internal hepatic glucose is generated by CaMKIIγLack damage and stimulated by constitutive activity CaMKII
For the function affect in assessment CaMKII in vivo hepatic glucose metabolism, WT mouse and Camk2g are checked-/-Mouse In fasting blood glucose level.It is consistent with vitro data, it observes compared with WT mouse, fasting Camk2g-/-Blood glucose water in mouse Flat appropriateness but statistically significant reduction (Figure 23 A).Compared with WT mouse, in knock-out mice, the difference of fasting glucose concentration is not Increase to Xun Huan insulin or Glucagon concentrations reduction is related (Figure 29 A, left side).Mutant mice is also shown in response to acetone Sour challenge test, plasma glucose are relatively low (Figure 23 B).It is consistent with primary HC data, fasting Camk2g-/-In the liver of mouse G6pc and Pck1mRNA is horizontal to have reduction (Figure 23 C).Similar data (figure is obtained in the mouse handled with glucagon 23D)。
It is consistent with these data, fasting blood-glucose (Figure 23 E) is reduced with gland-KD-CaMKII processing C57BL/6 mouse, it is described Processing makes liver CaMKII activity suppressions about 45% (Figure 29 B).As above, between control group and experimental group, Plasma glucagon (Figure 29 A, right sides) not different with insulin.It is consistent with blood glucose level data, in the mouse injected with KD-CaMKII, G6pc and The liver of Pck1mRNA expresses relatively low (Figure 23 F).Because CaMKII inhibits to reduce crucial glycogenolytic ferment G-6-Pase The level of mRNA, so check that acute and chronic CaMKII inhibits the influence to hepatic glycogen content, and as another finger of glycogen Mark also checks the percentage of periodic acid-schiff (PAS) positive cell.Data show gland-KD-CaMKII or CaMKII gene targets To the hepatic glycogen (Figure 23 G) increased in fasted mice.
Then mouse is handled by using gland-CA-CaMKII to check the effect of the constitutive activity liver CaMKII in mouse. CA-CaMKII groups have raised blood glucose level, increased liver G6pc and Pck1mRNA levels after pyruvic acid excitation and increase The hepatic glycogen content (Figure 29 C-E) added.CA-CaMKII applications do not change Plasma glucagon or insulin.These combinations Intra-body data shows that CaMKII influences plasma glucose levels, conversion of pyruvate into glucose and the table of hepatic glucose metabolic gene It reaches.
CaMKII promotes the nuclear location of FoxO1
A kind of central transcription factor involved in HGP is FoxO1, is mainly become by its positioning between cytoplasm and core Change regulation and control (Accili and Arden, 2004).Compared with Camk2g-/-Mouse measures transduction to from the separated HC of WT mouse The distribution of the FoxO1 of GFP marks.Under the conditions of serum starvation, in WT HC, most of GFP-FoxO1 are in core, and Camk2g-/-HC shows that GFP-FoxO1 is primarily located in cytosol (Figure 24 A).In addition, when HC gland-CA-CaMKII transduce When, FoxO1 becomes being mostly in core, and with gland-KD-CaMKII FoxO1 is caused to be mostly in cytoplasm (figure 24B).It should be noted that under " basis " cell culture condition used here, the core FoxO1 in the HC of culture be it is substantial amounts of, And the multiple increase therefore obtained with CA-CaMKII is limited.Therefore, using the of short duration incubation carried out together with insulin, substantially Core FoxO1 is lowered, and the incubation is then disclosed makes core FoxO1 dramatically increase (Figure 24 C) with CA-CaMKII.Although this reality Testing is carried out (referring to Figure 28 D) using high-caliber total CaMKII protein expressions, but uses the CA-CaMKII of relatively low MOI Similar experiment be shown under total CaMKII protein levels similar with endogenous CaMKII, core FoxO1 increase (Figure 30 A). To show interior dependency, the influence that the CaMKII γ tested in fasted mice lack or inhibit.In fasted condition, in liver Core FoxO1 is prominent (Figure 24 D, top trace), and in Camk2g-/-Mouse or with gland-KD-CaMKII transduction mouse in, It is substantially reduced (Figure 24 D, intermediate 2 traces).Feeding reduces core FoxO1, and under these conditions, core FoxO1 is by CA- Both CaMKII and glucagon increase (Figure 24 D, bottom trace).
In primary hepatocyte, the induction to G6pc mRNA of cAMP mediations is contained by Foxo1 shRNA>50%, so as to Instruction FoxO1 plays an important roll (Matsumoto etc., 2007) in endogenous environment.Consistent with these data, finding to work as makes During three shared FoxO binding sites mutation, the induction of the luciferase in people's G6PC promoters downstream is passivated (Ayala etc., 1999；Von Groote-Bidlingmaier etc., 2003) (Figure 30 B).However, this hepatoma cells system-report body structure Experiment is built not differentiate between cAMP effects and dexamethasone (dexamethasone) effect and endogenous situation cannot be accurately reflected.Example Such as, report that the strong degree of body induction induces much smaller (Matsumoto than the actual G6pc mRNA in primary hepatocyte herein Deng 2007), and the promoter element identified may not be for the unique site needed for regulating and controlling.Therefore, substitute and carry out this model The mode of the functional importance in the G6pc expression that assessment FoxO1 is mediated in CaMKII is further used as, it is thin to study primary liver The induction to endogenous G6pc in born of the same parents, and most importantly induction of the In vivo study to endogenous G6pc.To begin one's study, phase For lacking the L-Foxo1KO mouse of FoxO1 in liver, compare the energy of the G6pc in HC of the CA-CaMKII inductions from WT mouse Power (Matsumoto etc., 2007).With previous research (Puigserver etc., 2003；Matsumoto etc., 2007) unanimously, not In the case of FoxO1, the G6pc expression of forskolin induction is by containment (Figure 25 A, right figure).Most importantly by CA- The G6pc mRNA expression that CaMKII reaches increases lacks significantly passivation by FoxO1.In the case of there is no forskolin, such as institute It is expected that gene expression much lower (Figure 25 A, left figure；Pay attention to Y-axis scale), but the gene expression of even herein CA-CaMKII inductions Also it is almost completely dependent on FoxO1.Finally, it was demonstrated that the fact：Two kinds of other transcription factor CREB involved in HGP and The nuclear location of Crtc2 is not inhibited or lacked to reduce (Figure 30 C-D) by CaMKII.The data of these combinations promote with wherein CaMKII FoxO1 nuclear locations, it is consistent that this results then in the model that G6pc is induced.
The importance in HGP is damaged further to verify FoxO1 caused by being lacked or being inhibited as CaMKII, with containing phosphorus The adenoviral transduction for being acidified deficiency composing type core FoxO1 mutant (FoxO1-ADA) comes from Camk2g-/-The HC of mouse (Nakae etc., 2001).It observes that CaMKII γ lack to act on the G6pc and Pck1mRNA of forskolin the induction containment expressed to lead to It crosses and is transduceed with gland-FoxO1-ADA and be able to abolish (Figure 25 B).Pay attention to FoxO1-ADA used herein level it is sufficiently low so that The G6pc or Pck1 in the WTHC of forskolin processing, and Camk2g are not increased-/-The level of core FoxO1 in+ADA groups is similar to Endogenous level (illustration, Figure 25 B) in WT+LacZ groups.In view of find that the system genitale of FoxO1 is struck the low Pck1 that do not influence and induced (Nakae etc., 2002；Barthel etc., 2001), the Pck1 results in Figure 25 B and those in Figure 22-23 are the result is that cause Concern.However, the more violent silence of FoxO1 can contain Pck1 (Matsumoto etc., 2007), and therefore manipulation CaMKII can That environment can more be corresponded to.It is consistent with influences of the FoxO1-ADA to HGP gene expressions, at gland-FoxO1-ADA adenovirus Reason mouse has saved the damage (Figure 25 C-E) of the glucose homeostasis in the mouse of gland-KD-CaMKII processing.Pay attention to this As a result the type transduction of cannot be by being carried out in ADA groups with gland-KD the defects of is explained (illustration, Figure 25 E).In short, these results Promote the model of HGP consistent by promoting the nuclear location of FoxO1 with wherein CaMKII.
Effect in the FoxO1 nuclear locations that non-AKT- phosphoric acid FoxO1 sites and p38MAP kinases are mediated in CaMKII γ
Insulin/Akt promotes to arrange (Brunet outside FoxO1 cores by making T24, S253 and S316 (mouse residue) phosphorylation Deng 1999).Although CaMKII is a kind of kinases, it can activate phosphatase and be reduced from there through indirect at these sites Phosphorylation promote the nuclear location of FoxO1.However it has been found that from Camk2g-/-In the liver of mouse, at these three sites Phosphorylation do not change (Figure 31 A).Also the FoxO1 positioning in HC and active (Accili and Arden, 2004) can be influenced FoxO1 acetylations also lack from CaMKII influences (Figure 31 B).
FoxO1 also can be subject to phosphorylation (Asada at other Ser/Thr residues by other kinases of such as p38MAP kinases Deng 2007), and these phosphorylation events may promote FoxO1 nuclear locations rather than outer row.It is to assess CaMKII in non-Akt sites Phosphorylation in possibility effect, use the model shown in Figure 24 A, i.e. serum starvation WT HC and with also carrying FLAG labels GFP-FoxO1 transduction Camk2g-/-(KO)HC.Immunological purification is carried out to FoxO1 using anti-FLAG, then reduction, alkylation And the three protease schemes that use is developed by MacCoss etc. (MacCoss etc., 2002) carry out proteolytic digestion.Pass through TiO2Chromatograph enrichment Phosphorylated Peptide and the LC- then carried out by using MS bases shotgun (shotgun) proteomics method MS/MS (Cantin etc., 2007) and unmarked quantitative (Cantin etc., 2007) for being carried out by spectrum counting are analyzed.With About 70% sequential covering rate identifies FoxO1 protein, and identifies total 57 kinds of phosphoeptides of WT samples and KO samples 63 kinds of phosphoeptides are amounted to (in http:Table at //fields.scripps.edu/published/foxo1_Tabas_2012/ S1 and S2, the table are incorporated herein in its entirety by reference).Peptide false discovery rate (FDR) is less than 1 percentage.Using tight Lattice selection criterion will be so that the phosphoeptide of all identifications will all have high confidence level.
Using phosphoeptide analysis tool Debunker and Ascore (Lu etc., 2007；Beausoleil etc., 2006) further These criterions of verification make it possible to identify 11 phosphorylation sites：S284、S295、S326、S467、S475、T24、S246、 S253, S413, S415 and T553 (for mouse FoxO1 sequences, referring to Figure 31 C).Figure 26 A are shown from KO samples and WT samples The respective spectrum of this 11 kinds of FoxO1 phosphoeptides counts, Debunker score and A are scored.Most of phosphorylation sites are significantly larger than Respectively for the high confidence level cutoff value 0.5 and 12 of Debunker and Ascore.Make the value that Debunker scores or A scores slightly Relatively low 5 sites experience manual authentication, and their note tandem mass spectrum be shown in Figure 32 D-E (for KO peptides 4 and 5) and with On in the network address (for WT peptides 7,10 and 11) that refers to.The feature b ion and/or y ions in phosphoric acid site are all identified.Score compared with Low most probable, which is attributed to low abundance fragment ions or is lacked the neutrality of phosphoric acid due to the property of the amino acid sequence of these peptides, to be lost It loses.
The spectrum KO of the peptide calculating higher than 10 is counted only for the combination spectrum in KO samples and WT samples:WT is counted than using In the measurement of the relative expression for the Phosphorylated Peptide for obtaining identification.By this analysis, based on cutoff value<0.5, in KO, only The phosphorylation of S295, S467, S475 (peptide 2,4 and 5) are significantly lower, and wherein KO counts score compared with the spectrum of WT is not 0.45th, 0.38 and 0.5.Although there is the peptide containing p-S246 combination spectrum to count 7, and therefore be not up to>10 preassign Criterion, but compared with WT, it is shown in KO compared with low tendency (2 pair 5,0.4).On the contrary, S326 (peptide 3) has ratio 1.65, from And indicate compared with WT, it is raised in KO.
As relatively low some sites in KO Elementary Function test, using coding 7 Ser residues (including Ser295 and 475 and Ser246) at have S-A mutation FoxO1 (7A-FoxO1) available plasmid (Asada etc., 2007). When in similar level in Foxo1-/-When being transfected in HC, in response to glucagon, 7A-FoxO1 shows significantly less than WT The nuclear location of FoxO1, and with mutation FoxO1 transfection cell in cytoplasm FoxO1 higher (Figure 26 B).Also test have with Construct (mutant 9A) that 2 additional S-A in 7A in identical 7 S-A mutation plus S326 and S467 are mutated (Asada etc., 2007).This mutant shows similar deficiency nuclear location (Figure 31 F).In addition, although consistent with the data in Figure 24 D, gland- CA-CaMKII transductions increase core WT-FoxO1, but CA-CaMKII does not increase core 7A-FoxO1 (Figure 26 C).The data of these combinations Directly or indirectly change in a manner of promoting FoxO1 nuclear locations the phosphorylation of some Ser residues in FoxO1 with wherein CaMKII Model it is consistent.
Asada etc. (Asada etc., 2007) discoveries are in the HEK293T cells transfected with upstream p38 kinases MKK6, FoxO1 The evidence being phosphorylated at several sites including Ser284,295,467 and 475.Because p38 has involved the stimulation in HGP In (Cao etc., 2005), and when being studied in neuron, CaMKII can activating P 38 (Blanquet, 2000), it is contemplated that CaMKII → p38 → FoxO1 phosphorylations/nuclear location approach involved in HGP.It first confirms that in the liver of fasted mice, p38 quilts Phosphorylation (phosphorylation be p38 activation measurement) (Figure 32 A) and by high-affinity competitive inhibitor SB202190 inhibit P38 blocks the expression (Figure 32 B, left side) in the HC that G6pc and Pck1 is stimulated in glucagon.Using gland-Cre transduction from From P38afl/flThe separated HC of liver of mouse confirms inhibitor data (Figure 32 B, right side).Between test CaMKII and p38 Potential association is compared from WT mouse and Camk2g-/-Phosphorus in the HC that serum starvation HC and discovery the CaMKII γ of mouse lack Sour p38 is significantly reduced (Figure 32 C).Then determined by comparing the fasted mice handled with intermedium control and SB202190 Whether p38 is related in internal liver FoxO1 nuclear locations.Between 7 mouse in each group, (reflect p38 activity in p-MK2 P38 kinases target) basic horizontal with inhibited by SB202190 the aspect of degree two of MK2 phosphorylations there are a certain degree can Denaturation.Therefore, the core FoxO1 of all 14 mouse is drawn compared with p-MK2.Core FoxO1 in data display mouse is with p38 Inhibit and clearly reduce, i.e., as indicated by by relatively low p-MK2 (Figure 32 D).In addition, p38 inhibitor reduction glucagons Core GFP-FoxO1 (Figure 32 E) in the HC of processing.The data of these combinations are activated with wherein CaMKII by p38 promotes FoxO1 The model of nuclear location is consistent.Whether p38 is by directly making above-mentioned Ser residues (Asada etc., 2007) phosphorus in FoxO1 It is acidified to work in the FoxO1 nuclear locations induced in CaMKII still undetermined.
Effect in hepatic glucose metabolism of the CaMKII in obesity
Being partly due to glucagon facilitates obesity to neutralize it unbalanced HGP rises of insulin signal transduction Under its insulin resistant state hyperglycemia (Sorensen etc., 2006；Unger and Cherrington, 2012； Saltiel,2001).It is the effect during hepatic glucoses of the test CaMKII γ in obesity environment is metabolized, in two obesity Seek the evidence of liver CaMKII γ activation in mouse model.It was found that in ob/ob mouse and in a manner of high calorie diet high in fat Place the WT mouse (obesity of meals induction of 20 weeks；DIO) in the liver of the two, the level of p-CaMKII rather than total CaMKII are Significantly higher (Figure 27 A).The antibody specificity of both anti-p-CaMKII and anti-CaMKII in fat liver is by fat Camk2g-/- There is no Western blotting bands in mouse to show.Then by comparing the ob/ with gland-KD-CaMKII and gland-LacZ control transductions Fasting plasma glucose and the expression of liver FoxO1 target genes in ob mouse carry out testing functionality importance.With gland-KD- The mouse of CaMKII processing has relatively low fasting glucose, compared with hypoglycemia after pyruvic acid excitation and including G6pc and The relatively low expression (Figure 27 B-D) of three kinds of FoxO1 target genes of Pck1.The mouse that these variations are not handled with gland-KD-CaMKII In higher plasma insulin or be associated compared with low weight.Therefore, in the liver of obesity mice, liver CaMKII is activated and regulates and controls Hepatic glucose is metabolized and the expression of FoxO1 target genes.
It discusses
What the data in this report provided calcium mediation is the card of a part for the approach that can be outlined below to the regulation and control of HGP According to：Glucagon/fasting → cAMP/PKA → IP3R1 → Ca2+ I→ CaMKII → core FoxO1 → HGP.CaMKII is also mediated HGP rises (Figure 27) in obesity mice, and although needing more to study in this area, it is likely that be to drive herein Power be also glucagon (Sorensen etc., 2006；Unger and Cherrington, 2012；Saltiel,2001).Therefore, This result of study has influence for three basic areas relevant with HGP：Glucagon and fasting and obesity/pancreas islet Plain resistance stimulates the molecular mechanism that HGP is relied on；Molecule between intracellular Ca2+ and HGP associates；With the tune of FoxO1 nuclear translocations Control.It is especially interested in aftermentioned problem, because while FoxO1 phosphoric acid of the existing many reports on insulin/AKT- mediations Change and FoxO1 acetylations how to promote to arrange outside core FoxO1 (Lin and Accili, 2011；Van der Horst and Burgering, 2007), but the FoxO1 cores to occurring in the case of there is no insulin or in insulin resistance environment into Emphasizing for the regulation and control entered is less.
CaMKII approach is in the downstream of cAMP/PKA, and therefore natively supplement is stimulated other pancreas hyperglycaemia of HGP by it Element-PKA approach.So far, circumferential edge indicates that these other approach exist rather than also exist parallel with CaMKII approach The downstream of CaMKII.For example, glucagon-PKA directly makes cAMP response elements combine (CREB) protein phosphorylation, it is described Protein is by transcribing induction FoxO1 transcriptional coactivator PGC1 α (Herzig etc., 2001), but compared with KD-CaMKII mouse, Difference (Figure 30 B) is not present in core CREB in liver from gland-LacZ mouse.This is important result of study, because there is god Show that CaMKII can make the CREB in some environment live through the vitro data in member and in the RAW264.7 cells of RANKL processing Change/phosphorylation (Dash etc., 1991；Sheng etc., 1991；Ang etc., 2007).It has also been found that CaMKII shortages do not influence core Crtc2 (Figure 30 C), the Crtc2 are another transcription activators involved in HGP.CaMKII and common reality in these data instruction liver Show a series of nuclear location of appropriate transcription factors so that these other approach of HGP to be mediated to work parallel.Situation on Crtc2 Especially cause concern, because the IP3R activation of glucagon/PKA mediations and ER calcium are released through another calcium sensitivity enzyme calcium tune Neural phosphatase promotes Crtc2 nuclear locations.In fact it has been found that for the G6pc mRNA of containment forskolin induction, inhibit CaMKII and calcineurin are additive.Therefore, common near end signal pathway causes two kinds of key HGP transcriptions The core of the factor Crtc2 and FoxO1 enter by different distal end mechanism coordinations.In this regard, what is interesting is notice that previous research show Show that the drug that calcium is promoted to enter through plasma membrane actually reduces the Pck1mRNA (Valera etc., 1993) in HC, this may indicate that calcium It is to determine the factor of downstream events into the approach in cytoplasm.
FoxO1 passes through Akt phosphoric acid at Thr24, Ser253 and Ser316 (mouse sequence number) by insulin/growth factor Change to promote to arrange outside its core.CaMKII promotes FoxO1 nuclear locations, but under without being bound by theory, CaMKII, which can be activated, makes this The phosphatase of a little sites dephosphorylation.However, CaMKII γ shortages do not influence the phosphorylation of these three residues, and it is nor affected on FoxO1 acetylations (Figure 31 A-B).On the contrary, find that evidence suggests the phosphoric acid of other Ser residues on CaMKII mediations FoxO1 Change, and the experiment of Ser-Ala FoxO1 mutant indicates to work in the FoxO1 nuclear locations for acting on CaMKII mediations.
Associating between CaMKII and FoxO1 phosphorylations can be directly or indirect.Indirect mechanism, i.e. CaMKII lives Change the mechanism that another kinases is relied on, the mode that FoxO cores retain can be promoted to make on non-Akt sites with other kinases The previous research result of FoxO phosphorylations be associated (Essers etc., 2004；Chiacchiera and Simone, 2010).Based on this The research of literary p38 inhibitor and gene target data and Asada etc. (Asada etc., 2007), under without being bound by theory, P38MAPK is also able to carry out this function, and may actually be the mediators of the FoxO1 nuclear locations of CaMKII inductions.Support this A be between CaMKII and p38 and between p38 and HGP there are associated report (Cao etc., 2005；Blanquet, 2000).Although showing that p38 makes FoxO1 phosphorylations and thus activates FoxO1 there is no positive evidence, glucocorticoid promotes The ability of FoxO1 nuclear locations in rat myocardial cell is related to activation/phosphorylation of core p38, and immunofluorescence microscopy It can be interacted with each other (Puthanveetil etc., 2010) with IP/ immunoblotting datas instruction p-P38 and FoxO1.Cause concern Be the p38 (Naimi etc., 2007) that can be activated evidence suggests FoxO1 in HC, and it is therefore possible to be FoxO1-p38 Feedforward approach can expand effect of the CaMKII-p38 approach indicated herein to FoxO1 nuclear locations.However, it is necessary to more research comes It determines the effect of p38 and furthers elucidate the mechanism that CaMKII promotes FoxO1 nuclear locations to be relied on.
It was found that effects of the calcium-CaMKII in HGP not only provides the understanding of the physiological defense to being directed to fasting hypoglycemia, and And it can also disclose for the therapeutic targets for being disturbed glucose metabolism being present in insulin resistance environment, such as by Figure 27 Data indicated by.In fact, in diabetes B, out-of-proportion HGP and glucagon are passed compared with insulin signaling The imbalance led facilitate hyperglycemia (Sorensen etc., 2006；Saltiel,2001).In addition, glucagon signal passes It leads and also involves in type 1 diabetes (Unger and Cherrington, 2012).In this scenario, future studies will be further It illustrates pathophysiological role and mechanism of the liver CaMKII γ in obesity, insulin resistance and diabetes and thus assesses it and make For the potentiality of the therapeutic targets in these illnesss.
Experimental arrangement
The measurement of CaMKII activity
According to manufacturer specification, CaMKII activity is measured using the CaMKII assay kits from Promega.Such as It is burnt by being exposed to the 50mM HEPES for containing 1% triton-X100,150mM NaCl, 10mM in legend after indicated processing HC Sodium phosphate, 10mMEDTA, 10mM EGTA, 1mM Na3VO4, 50mM NaF, 1mM PMSF and 5 μ g ml-1Leupeptin comes for 5 minutes Dissolve them.Then, incite somebody to action [γ-32P] ATP and biotinylation CaMKII peptide substrates are added to lysate or immune precipitation (see below) in compound.After being incubated 10 minutes at 30 DEG C, using SAM biotins trapping film make [32P]-phosphorylation bottom Object with it is remaining [32P] ATP separation and then quantified using scintillation counter.It is measured under ± calmodulin, and comfortable presence The activity value obtained in the case of calmodulin subtracts the activity value in the case of there is no calmodulin.
Glucose in primary HC generates
Such as described progress glucose, which generates, measures (Yoon, 2001).In brief, collect and cultivate original as described above After mouse HC, by cell culture medium be converted into being supplemented with 20mM sodium lactates and 2mM Sodium Pyruvates without glucose and phenol DMEM (pH7.4).After when culture 16 is small, 500 μ l culture mediums are collected, and use colorimetric Glucose estimation kit (Abcam) glucose content is measured.Then standardize reading compared with the total protein concentration in whole cell lysate.
Mouse experiment
Camk2g is generated as discussed previously-/-Mouse (Backs etc., 2010) and hybridization in C57BL6/J backgrounds.ob/ob Mouse obtains from Jackson laboratories.For the experiment in Figure 27, the height of mouse standard diet or 60%kcal from fat Fatty diet feeding, and according to 12 it is small when fight-darkness cycle supported.Recombined adhenovirus is delivered by tail vein injections (1.5×109A plaque formation unit/mouse), and started to test after 5 days.Use Glucose meter (One Touch Ultra, Lifescan) measurement freely obtain underwater fasting 12-14 it is small when mouse in fasting blood-glucose.When fasting 17 is small Afterwards, with injection 2g kg in peritonaeum-1The pyruvic acid of weight carries out pyruvic acid tolerance test.After subsequent 2 it is small when measure blood Sugar level.By in 10pmol g-1Dosage under continue to carry out for 4 days injection in daily peritonaeum to apply smooth Spongistatin to mouse C.P38a is generated as discussed previouslyfl/flMouse (Engel etc., 2005).
Hepatic glycogen measures
50-100mg is made to freeze liver in the 1ml H with protease and inhibitors of phosphatases2It homogenizes in O.Then sample is made Sheet and KOH (1:2) mix, boil 25 minutes and washed with 70% ethyl alcohol.It is drying precipitated and be dissolved in 100 μ l H2In O, And according to manufacturer specification, glycogen content is assessed using glycogen assay kit (Abcam).Data represent average value ± SEM.
The PAS dyeing of mouse liver slice
By liver sample be fixed in 10% neutral buffered formalin 24 it is small when and be embedded in paraffin.It is said according to manufacturer Bright book uses the glycogen in periodic acid-schiff (PAS) coloring agent (Sigma) stained slice (5 microns).Then with hematoxylin pair It is checked than stained slice and by optical microscopy.For quantifying for PAS dyeing, random selection is from 4 different sections 5 regions, and the number of PAS positive cells is counted and is expressed as the percentage (Hammad etc., 1982) of total number of cells.It is right Unwitting 2 independent investigators of identity of sample are analyzed.
The analysis of mass spectrometric data
Using ProLuCID, DTASelect2, Census, DeBunker and Ascore, with integrated proteomics flowing water Line-IP2 (Integrated Proteomics Applications, Inc., San Diego, CA.http:// Www.integratedproteomics.com/ protein and phosphoeptide identification, quantitative and analysis of Phosphorylation) are carried out.It uses RawExtract1.9.9(http://fields.scripps.edu/downloads.php) by spectrum original document from original File is extracted into ms1 and ms2 files (McDonald etc., 2004), and compared with EBI IPI murine protein matter databases (http://www.ebi.ac.uk/IPI/IPImouse.html, issue on March 24th, 2010) search tandem mass spectrum.To be accurate Estimate peptide probability and false discovery rate, use the bait data of the reversed sequence containing all proteins for investing target database Storehouse (Peng etc., 2003).Using ProLuCID (Xu etc., 2006) algorithm, make tandem mass spectrum and sequence under 50ppm peptide quality tolerances Row matching.It is to carry out ProLuCID searches on the Intel Xeon clusters run under (SuSE) Linux OS.Search space includes Belong to all complete and half portion tryptic peptide candidates in quality tolerance window.Cysteine ureidomethy (+ 57.02146Da) it is considered as static modifying, and be considered as can for the phosphorylation (+79.9663) on serine, threonine and Tyrosine Become modification.
The parameter determined using two kinds of SEQUEST (Eng etc., 1994), i.e. cross correlation score (XCorr) and cross correlation The normalized difference (δ CN) of score, DTASelect (Tabb etc., 2002；Cociorva etc., 2007) assessment peptide/spectrum in Correctness with (PSM).By search result according to state of charge (+1 ,+2 ,+3 and more than+3) and trypsase state (complete pancreas Protease, half portion trypsase and non-trypsase) grouping, so as to generate 12 different subgroups.In these subgroups Each in, obtain (a) directly and distribution of Xcorr, δ CN and δ mass values of (b) bait data storehouse PSM, and then pass through Discriminant analysis separates direct subgroup and bait subgroup.It is generally impossible to completely separable direct PSM subgroups and bait PSM subgroups；Cause This, peptide matching probability is calculated based on the Nonparametric Fitting Regression of direct and bait score distribution.Peptide confidence level 99.5% is arranged to Minimum threshold, and only receive the phosphoeptide that δ mass is less than 10ppm.False discovery rate is calculated as that bait PSM is reversed to pass through Percentage in all PSM of 99.5% confidence level threshold value.After this last filtration step, estimation protein mistake hair Now rate is both less than 0.1% with peptide false discovery rate.After database search and DTASelect2 filterings, with use The IP2 analysis of Phosphorylation tool analysis phosphoeptides of Ascore (Beausoleil etc., 2006) and Debunker (Lu etc., 2007). Use spectrum method of counting quantitation of peptides and phosphoeptide (Liu et al., 2004).
Statistical analysis
All results are all rendered as average value ± SEM.Using student t (student's t-test) is examined to calculate normal state point The P values of cloth data and the P values that non-normal data is calculated using graceful-Whitney (Mann-Whitney) rank sum test.
Appended experimental program
Reagent and antibody
Glucagon, pyruvic acid, forskolin, H89 and 8- bromo- cAMP and SB202190 come from Sigma.BAPTA-AM and Anti- phosphocarnic acid albumen (Np) antibody comes from Invitrogen.Smooth Spongistatin C comes from EMD Chemicals.Anti- phosphoric acid Thr287CaMKII antibody comes from Imgenex and Novus；Anti- total CaMKII, anti-FoxO1 and anti-Ac-FoxO1 antibody come from Santa Cruz Biotechnology Inc.Anti- beta-actin and anti-phosphoric acid S316FoxO1 antibody come from Abcam.Anti- phosphorus Sour p38, anti-phosphoric acid MK2, anti-phosphoric acid CREB, anti-p38, anti-MK2, anti-CREB, anti-phosphoric acid T24FoxO1, anti-phosphoric acid S253FoxO1, Anti- HA and anti-FLAG antibody come from Cell Signaling.Anti- CRTC2 antibody is the present of doctor MarcMontminy.Previously retouch Stated coding LacZ, CA-CaMKII, KD-CaMKII, GFP-FoxO1 and Cre adenovirus (Pfleiderer etc., 2004； Tanaka etc., 2009；Akagi etc., 1997) and expanded by Viraquest, Inc. (North Liberty, IA).Such as the structure Build the plasmid (Asada etc., 2007) of coding FoxO1 mutant 7A and 9A.
Primary hepatocyte
Primary mouse HC is to separate (Matsumoto etc., 2002) from 8 to 12 week old mouse as discussed previously.For most Number experiment, by being incubated that HC is made to be subjected to staying overnight serum exhaustion in the culture medium containing 0.5% hyclone, and then in nothing When incubation 5 is small in blood serum medium, wherein indivedual process is indicated in legend.When after inoculation 12 is small, adenovirus construct is used Transduce HC, and is tested when after transduction 24 is small.According to manufacturer specification, jetPEI is usedTM- liver cell DNA is transfected Reagent (Polyplus-transfection, Inc.) transfected with WT, 7A- and 9A-Foxo1.
Immune precipitation
By be exposed to contain the 50mM HEPES of 1% triton-X, 150mM NaCl, 10mM sodium pyrophosphates, 10mM EDTA, 10mM EGTA, 1mM Na3VO4,50mMNaF, 1mM PMSF and 5 μ g/ml leupeptins 5 minutes make cell dissolution.With containing The dissolving buffer solution of 0.3-0.6 μ g antibody and 80 μ l agarose beads makes lysate (500 μ g proteins) reach total volume 1ml.When rotated mixture 14 is small in 1.5ml microcentrifugal tubes at 4 DEG C.It is immune to collect by being centrifuged under 16,000g Compound and with freezing dissolving buffer solution wash 3 times.
Ip3r1floxThe generation of mouse
It is following to generate Ip3r1floxMouse species：RP24- is cloned by using the BAC of the segment containing chromosome 6 245H16 (CHORI) carries out recombined engineering transformation to generate targeting construc, and the segment covers from C57Bl/6J mouse IP3The exons 1 of R1 to 9 (Liu et al., 2003；Warming etc., 2005).Plasmid pL451, pL452 and pL253 be by Doctor NealCopeland (NCI, NIH) friendship provides.In brief, Frt-Neo-Frt-loxP boxes (are come from into plasmid PL451 the upstream of extron 4 (the second encoded exon)) is inserted into, the flp restructuring then induced by agarose removes Neo boxes. By be inserted into loxP-Neo-loxP boxes (from carrier pL452) by second and the 3rd loxP sites introduce under extron 4 Trip.Finally, by the further downstream of the 3rd loxP sites of DTA boxes (coming from plasmid pL253) insertion containing thymidine kinase.All centres BAC constructs and final construct are all screened by PCR, and final construct carries out " fingerprint point by Acc65I digestion Analysis " simultaneously tests loxP functionality.Confirm crucial bond site by being sequenced.The BAC electroporations that gained is modified are thin to chimeric ES In born of the same parents' (CSL3 cell lines come from 129S6/SvEvTac mouse species).ES cell infusions will correctly be recombinated to C57BL/6 blastocysts In the mice embryonic in stage.Make chimeric males mouse with the breeding of C57BL/6 female mices to establish crosses.System genitale is transferred Generate Ip3r13xfloxMouse, and female is made wherein to eliminate floxed Neo boxes with EIIa-cre male hybridization to generate Ip3r1floxMouse.These mouse is then made to remove EIIa-cre allele with C57BL/6J mouse hybrids to educate.Pass through heterozygosis Breeding obtains final Ip3r1fl/flStrain.
Western blotting and RT-qPCR
Western blotting is carried out as discussed previously and RT-qPCR measures (Timmins etc., 2009).Use RNeasy kits (Qiagen) total serum IgE is extracted from HC.It is synthesized using oligomerization (dT) and Superscript II (Invitrogen) from 2 μ g total serum IgEs cDNA.According to manufacturer specification, carry out extracting core from liver using the core extracts kit from Panomics.On anti- Thr287-p and total CaMKII Western blottings are generally observed 2 bands being not present in the HC lacked in CaMKII γ and have When 3 bands.Their source is that alternative splicing or posttranslational modification are still undetermined.
The mass spectroscopy of FoxO1 phosphoeptides
In MOI WT mouse and Camk2g are come from gland-FLAG-FoxO1 transductions for 2 times-/-The HC of mouse.It is subjected to cell When overnight serum is exhausted and then incubation 5 is small in serum free medium.Immunological purification is carried out to FoxO1 using anti-FLAG.It is logical The sample solution (cold) of 1 volume of mixing is crossed with 100% (w/v) TCA (6.1N, Sigma) of 1/3 volume to make to delay in ice-cold Tris Rush the FLAG-FoxO1 precipitations in physiological saline.After when on ice 3 is small, sample is centrifuged at 4 DEG C 30 minutes, and suction out supernatant Liquid, so as to leave about 5-10 μ l in pipe so as to not disturb precipitation.Precipitation is washed with ice-cold acetone twice (500 μ l every time). After each washing, solution is centrifuged 10 minutes.Then dry final precipitation 1-2 minutes on Speed-vac.
Peptide (Delahunty and Yates, III, 2005 are generated by such as described progress proteolysis；MacCoss etc., 2002).TCA is precipitated and dissolved in the 100mMTris-HCl (pH8.5) that 60 μ l contain 8M ureas, and then by adding 500mM Three (2- carboxyethyls) phosphines (TCEP) go back crude protein up to ultimate density 5mM.It is incubated at room temperature after 20 minutes, by adding Add 500mM iodoacetamides makes cysteine residues carboxy methylation to reach ultimate density 10mM.It incubates in the dark at room temperature Educate solution 30 minutes and then decile into 3 pipes.In a pipe, then by adding the 100mM Tris- of equal volume HCl (pH8.5) is by 2 times of the concentration dilution (to 4M) of urea, and then about 1:100 enzymes:Substrate ratio (wt:Wt withered grass is added under) Bacillus protease (subtilisin) (Promega) and be incubated in the dark at 37 DEG C 4 it is small when.By other 2 Sample Dilutions 4 Again (to 2M), and about 1:100 enzymes:Substrate ratio (wt:Wt elastoser (elastase) and trypsase are added under) (Promega), and then 2 sample incubations are stayed overnight in the dark at 37 DEG C.Gained peptide from 3 digestive juices is merged Into a pipe and it is dissolved in 90% formic acid to obtain the ultimate density 2% in 10% acetonitrile.In TiO2 enrichments and LC- Before MS/MS analyses, sample is stored at -20 DEG C.
TiO2 enrichments such as are carried out to phosphoeptide by Cantin (Cantin etc., 2007) is described.By by TiO2 (5 μ Partisphere, Whatman, Clifton, NJ) it is filled in a manner of pressure homogenate to (250 μm of fused silica capillary I.d. until 5cm length is to prepare TiO2 columns in), and peptide mixer pressure is loaded on column.Column buffer solution A and B (for Buffer solution forms, referring to sections below) continuously washing, and then directly being washed phosphoeptide using 250 mM ammonium hydrogen carbonate (pH 9) Take off to 55 μm of reverse phases of cm 100 μm of i.d. Kasil sintering end packed column (Gemini C18, Phenomenex, Torrance, CA) in, the column is connected to the drawing tip (pulled-tip) point of the bed volume with the identical reverse phases of 15cm Analyse column.Second column connects to carry out mass spectroscopy analysis with Agilent1200 quaternarys HPLC pumps (Palo Alto, CA).
HPLC buffer solutions used are water/acetonitrile/formic acid (95 as buffer solution A:5:0.1, v/v/v) and as buffering Water/acetonitrile/formic acid (20 of liquid B:80:0.1, v/v/v).Gradient is as follows：10 minutes 100% buffer solution As, 5 minutes from 0 to The gradient of 15% buffer solution B, 65 minutes gradients from 15 to 45% buffer solution Bs, 15 minutes ladders from 45 to 100% buffer solution Bs Degree and 5 minutes 100% buffer solution Bs.With LTQ-Velos-Orbitrap mass spectrographs (ThermoFisher, San Jose, CA) Data dependency tandem mass spectrum measure (MS/MS) is carried out to analyze.It will be eluted under 2.5 kV spray voltages of application distal end from LC columns The direct electron spray of peptide into mass spectrograph.The cycling of a complete scan MS spectrum (m/z 300-1800) is obtained, is followed by 20 of related the 1st to the 20th most strong ion selected from complete MS spectrum sequentially generated under 35% normalized collision energy MS/MS events.For MS scannings scan the two with MS/MS, the number of micro- scanning is 1, and during maximum ion injection Between be 25 ms and 50 ms respectively.Dynamic used excludes to set as follows：Repeat count, 1；The repetition duration, 30 seconds；It excludes clear Single size, 500；With exclude the duration, 120 seconds.MS scan functions and HPLC Solvent Gradients are by Xcalibur data systems (ThermoFisher) control.
G6PC promoters-luciferase assay
FAO liver cells are blended in the construct of the nucleotide -1227 to+57 of people's G6PC promoters of luciferase with coding (- 1227 WT) or identical construct (- 1227 Mut) transfection of 3 shared FoxO binding sites mutation, the binding site It is -187 to -183 (GTTT → CGAG)；-171(G → C)；With -164 (A → C), so as to destroy in G6PC promoters Shared IRS sequences T (G/A) TTT (Ayala etc., 1999；Von Groote-Bidlingmaier etc., 2003).In position -164 The end mutation at place is in cAMP response elements (CRE；- 161 to -152) upstream so that CRE it is unaffected (Barthel etc., 2001).With the processing liver cell 16 of the serum free medium with 1%BSA containing 0.1 mM cAMP and 1 μM of dexamethasone it is small when, Then dissolving and analysis of fluorescence element enzymatic activity.Luciferase units (RLU) are standardized compared with the untreated cell in each group.
Embodiment 9：InsP3 receptor modulators are in hepatic gluconeogenic when fasting and diabetes
In the fasted state, cycling glucagon increases by inducing gluconeogenesis program that hepatic glucose is promoted to generate. CAMP approach is triggered by the way that the sub- CRTC2 dephosphorylations of CREB co-activatings is made to increase gluconeogenesis gene expression (1).Glucagon portion Ground is divided to pass through the inhibition to CRTC2 kinases SIK2 of PKA mediations to promote CRTC2 dephosphorylations.Many Ser/Thr phosphatases seem It can make CRTC2 dephosphorylations (2,3), but the mechanism that hormone signal regulates and controls these enzyme institute foundations is still indefinite.It is shown here to be Glucagon is by transferring intracellular Ca2+ storage cavern and activation calcium/calmodulin-dependent Ser/Thr phosphatase calcium tune nerve phosphorus Sour enzyme/PP2B carrys out the CRTC2 dephosphorylations in cell cultured supernatant.The 1,4,5- InsP3s that glucagon is mediated by PKA The phosphorylation of receptor (InsP3R) increases kytoplasm liquid calcium, associated with CRTC2 shown here as this.After InsP3R activation, InsP3R enhances gluconeogenesis gene expression by promoting the CRTC2 dephosphorylations of Role of calcineurin in signal transduction.During feeding, Insulin signal transduction, which increases, to be inactivated to reduce CRTC2 activity by the InsP3R of AKT mediations.In diabetes, InsP3R lives Property increase, so as to cause gluconeogenesis program raise.Because liver InsP3R and calcineurin, which are lowered, improves insulin resistance In circulating-glucose levels, so these results illustrate under InsP3 regulation fasted conditions and diabetes in liver Portugal In the level that grape sugar generates, how to influence each other between cAMP and calcium approach.
As a result and discuss
Testing a series of Ser/Thr protein phosphatases (PP) inhibitor blocks the CRTC2 in response to glucagon to live The ability of change.Make the CRTC2 of glucagon induction exposed to PP2B/ Calcineurin inhibitors cyclosporin A (CsA) Dephosphorylation and core transposition are damaged, but the okadaic acid (okadaic acid, OA) of the inhibitor as PP1, PP2A and PP4 is not So (Figure 33 A, Figure 37 A).CsA and other Calcineurin inhibitors also reduce CRE- luciferases (luc) report body Active (Figure 33 A, Figure 37 B), but they are in expression phosphorylation deficiency (Ser171,275Ala) form and therefore activity form Without effect (Figure 37 C-E) in the cell of CRTC2.
Based on the ability of CsA interference CRTC2 activation, consideration is whether calcineurin can promote in response to pancreas height The CRTC2 dephosphorylations of blood glucose element.Support this idea be CRTC2 contain 2 mediation with calcineurin associate be total to There is (PXIXIT) primitive (3,4) (Figure 38 A, B).In addition, two primitives of mutation make the glucagon dependence dephosphorylation of CRTC2 It is damaged (Figure 33 B) and prevents its core transposition (Figure 38 C), thus lower CRE-luc activation (Figure 33 B).
Based on these as a result, whether test calcineurin adjusts the expression of gluconeogenesis program.In liver cell, gland Virus overexpression calcineurin catalytic subunit makes the CRTC2 dephosphorylations in response to glucagon, CRE-luc live Property and glucose production strengthen, although calcineurin strike it is low have adverse effect (Figure 33 C).Although calcium tune nerve phosphoric acid Enzyme can adjust CRTC2 activity, but the excessive table of calcineurin indirectly by the effect to cAMP signal transductions in principle Reach or strike the phosphorylation (Figure 38 D) of the low cell PKA substrates not changed in the cell of glucagon.
Then check whether calcineurin adjusts internal hepatic gluconeogenic.(2 times) overexpression calcium of appropriateness in liver Adjust neural phosphatase that gluconeogenesis gene expression, liver CRE-luc activity and fasting plasma glucose concentration is made to increase (Figure 33 D, Figure 39 A).Phase Instead, striking low liver calcineurin reduces the expression of gluconeogenesis program and circulating-glucose levels is made to reduce (Figure 33 D, figure 39B), so as to illustrate the phosphatase promote liver in fasting adapt to.Calcineurin seems through CREB pathway stimulations sugar Heteroplasia；Exhaust that CRTC2 makes the effect of calcineurin overexpression in this environment by blocking (Figure 40).
Recognize that Calcineurin Activities increase dependent on intracellular Ca2+, so whether test cAMP approach stimulates calcium It is mobile.Primary hepatocyte is made quickly to increase (Figure 34 A, Figure 41 A) exposed to glucagon triggering cell free calcium；By using PKA inhibitor H89 coprocessing, these effects are able to part and reverse (Figure 41 B).Intracellular Ca2+ rise seems Crucial, because being incubated CRTC2 dephosphorylations and the CRE-luc activation made in response to glucagon altogether with calcium chelating agent BAPTA It is damaged (Figure 34 B).The effect of cAMP signal transductions is not blocked exposed to BAPTA in response to pancreas hyperglycaemia by contrast with calcium The CREB phosphorylations of the PKA mediations of element.
Under without being bound by theory, cAMP can be moved by calcium channel phosphorylation in PKA dependent cells to increase calcium. To identify in response to glucagon, it is subjected in being studied by the mass spectroscopy of the PKA protein for carrying out phosphorylation, from phosphoric acid The sero-fast immune precipitation recycling 1,4,5- inositol triphosphate receptors 1 (InsP3R1) (Figure 41 C) of PKA substrates.InsP3R1 and it Associated families member (InsP3R2, InsP3R3) be they in response to extracellular signal activate after, promote endoplasmic reticulum (ER) calcium release channel (5-9) of calcium storage cavern movement.In addition, the phosphorylation that cAMP agonists can be mediated by PKA is also shown To enhance InsP3R receptor actives.
By making liver cell exposed to smooth Spongistatin C (Xc) or inhibiting InsP3R by striking low all 3 kinds of InsP3R Make to be damaged in response to the movement of the kytoplasm liquid calcium of glucagon and forskolin and calcineurin activation and (Figure 34 A, scheme 42A).In addition, Xc processing and InsP3R, which strike low also blocking glucagon, activates and induces sugar to CRTC2 dephosphorylations, CRE-luc The effect (Figure 34 C, Figure 42 A, B) of heteroplasia program.Confirm InsP3R consumptions using the liver cell (10) from InsP3R2 knock-out mices The influence (Figure 42 C-E) exhausted, the InsP3R2 are the main InsP3R hypotypes in these cells.
Based on these as a result, under without being bound by theory, also fasting glucose generates InsP3R in adjustable body.By striking All 3 kinds of Insp3R in low liver destroy InsP3R2 genes and make fasting CRE-luc to reduce liver InsP3R expression by targeting Activity, gluconeogenesis gene expression and circulating glucose concentration reduce, so as to illustrate these receptors in glucose homeostasis Importance (Figure 34 D, Figure 43).
Whether active by the phosphorylated regulation InsP3R of PKA mediations test glucagon.According to phosphoric acid PKA substrates Antiserum carry out Western blotting measure, make liver cell exposed to glucagon make InsP3R1 and InsP3R2 and The phosphorylation of InsP3R3 increases；These effects block (Figure 35 A, Figure 44 A) by PKA inhibitor H89.In addition, in InsP3R1 Shared PKA sites at serine residue (Ser1589, Ser1756) be mutated into alanine and make in response to glucagon InsP3R1 phosphorylations are damaged (Figure 35 B) completely.Therefore, PKA deficiencies (S1589,1756A) InsP3R1 interference is over-expressed Calcium moves and calcineurin activation, and this reduces CRE-luc activation and glucose from exposed to glucagon The secretion (Figure 35 B-D) of liver cell.
Similar with glucagon, fasting also stimulates the liver InsP3R1 phosphorylations (figure at Ser1589 and Ser1756 44B).And overexpression PKA deficiencies InsP3R1 reduces fasting CRE-luc inductions, calcineurin activation and gluconeogenesis Gene expression, it is relatively low (Figure 35 E, Figure 44 C, D) so as to cause circulating glucose concentration.In short, these results support PKA mediations Important function of the InsP3R phosphorylations in hepatic gluconeogenic.
Under without being bound by theory, it can be important for its activation that CRTC2, which is adjacent to Ca2+ oscillations conduction mechanism,.It supports This opinion be find co-immunoprecipitation measure in, CRTC2 by its N-terminal CREB binding structural domains (CBD) with InsP3R1 associates (Figure 35 F, Figure 45 A-D).In addition, CRTC2 is rich in high density microsome (HDM) part containing ER, institute It states part and also contains InsP3R (Figure 45 E).InsP3R:CRTC2 associations seem that the CRTC2 positioning in perinuclear space be crucial Because RNA mediation InsP3R strike it is low cause CRTC2 redistribute in cytoplasm (Figure 45 F).By lacking in CRTC2 CBD makes CRTC2 target the N-terminal myristoylation signal of plasma membrane to destroy CRTC2 by addition:InsP3R interactions make sound In the CRTC2 dephosphorylations and CRE- of glucagon body activation should be reported by blocking (Figure 45 G-I).In short, these results indicate CRTC2 and InsP3R associations enhance its sensibility to fasting signal.
Under the conditions of feeding, inhibit gluconeogenesis by increasing CRTC2 phosphorylations insulin portion.Checking insulin is The effect that no interference InsP3R activates CRTC2.Support this idea is to have shown AKT by making InsP3R in Ser2682 At (in InsP3R1) phosphorylation come block calcium move (11).In fact, exempted from according to what is carried out with phosphoric acid AKT substrates antiserum Epidemic disease engram analysis make liver cell exposed to insulin InsP3R phosphorylations be made to increase (Figure 46 A)；Make Ser2682 (in InsP3R1 In) be mutated into alanine and blocked these effects.Insulin processing also reduces the pancreas in the cell of expression wild type InsP3R1 The movement of glucagons dependence calcium increases and calcineurin activation, but this is expressing AKT deficiencies (S2682A) Without effect (Figure 46 B) in the cell of InsP3R1.Therefore, it is thin in the liver of expression (S2682A)-InsP3R compared to wild type In born of the same parents, CRTC2 dephosphorylations, CRE-luc activity and glucose output rise (Figure 46 C).
Then check whether InsP3R1 is important in regulating and controlling internal hepatic glucose and generating by AKT phosphorylations.Feeding increases Liver InsP3R1 phosphorylations (Figure 44 B) at Ser2682.In addition, overexpression AKT deficiencies InsP3R1 part containment feedings lure The CRE-luc activity led reduces and gluconeogenesis gene expression, and (Figure 46 D) is raised so as to cause circulating glucose concentration.In short, this A little result instruction InsP3R phosphorylations that AKT is mediated during feeding are by blocking calcineurin dependence CRTC2 to take off Phosphoric acid inhibits hepatic gluconeogenic.
Check whether liver InsP3R signal transductions promote the gluconeogenesis in insulin resistance environment to increase.In ob/ob glycosurias In both sick animal and db/db diabetic animals, liver Calcineurin Activities enhance, so as to cause CRE-luc activity Increase (Figure 36 A, Figure 47 A, B).Point out the effect of InsP3R is the liver PKA phosphorylation activity in these diabetic mices The amount of InsP3R increases, and the amount of the nonactive InsP3R of AKT phosphorylations reduces (Figure 36 B).Correspondingly, strike in low db/db mouse Calcineurin or InsP3R CRE-luc activity, gluconeogenesis gene expression and hepatic gluconeogenic is made to reduce (Figure 36 C, figure 47C)。
In short, this paper results illustrate that glucagon is increased by the cytoplasm calcium that triggering causes calcineurin to activate Calais promotes the CRTC2 dephosphorylations (Figure 48) during fasting.Glucagon can by PKA mediate InsP3R phosphorylations come In liver and perhaps Ca2+ oscillations conduction explanation is increased in other insulin-sensitive tissues, between cAMP and Ca2+ oscillations pathway In the presence of for reaching interactional important regulating and controlling node.Calcium, which enters, to be inhibited to also indicate that presence can be sent out by PKA inhibitor H89 parts Wave the other regulation and control input (12,13) for the effect for increasing InsP3R activity together with PKA in response to glucagon.It has also been found that CRTC2 stimulates metabolic gene table by raising the sub- PGC1 α of nuclear hormone receptor co-activating in liver (14,15) and muscle (16) It reaches.Play an important role of to be fully realized in PGC1 α dependent transcriptions based on Ca2+ oscillations conduction, InsP3R also can be in this environment In play an important role.
Method
Adenovirus (17) is delivered by tail vein injections.Use IVIS imaging systems observation liver CRE-luc activity. 3-5 days after injection CRE-luc adenovirus, mouse is imaged.It is injected to overnight fasting and in pyruvic acid (2g kg-1) peritonaeum Mouse carry out pyruvic acid tolerance test.Insp3r2 knock-out mices (10) have been described.As report prepare culture it is primary small Hepatocytes (18).Cell grade Separation Research (18) is carried out using Primary mouse liver cell.Using CCD camera to The primary hepatocyte that fura-2 dyestuffs load carries out calcium imaging experiment.To the CRTC2 prepared from HEK293T cells be immunized precipitation and Mass spectroscopy is carried out from the sero-fast immune precipitation of the phosphoric acid PKA substrates of the primary hepatocyte preparation exposed to glucagon to grind Study carefully.Anti- InsP3R1 (A302-158A) and InsP3R3 (A302-160A) antibody are bought from Bethyl laboratories, anti-InsP3R2 (ab77838) antiserum comes from Abcam, and anti-calcineurin (610260) comes from BD Biosciences, anti-GRP78 (ADISPA-826-F) from EnzoLife Sciences, anti-phosphoric acid PKA substrates (RRXS/T, 9624), anti-phosphoric acid AKT substrates (RXXS/T, 9614) and CRTC2 (pS171,2892) come from Cell Signaling.Phosphoric acid (Ser275) is used as described CRTC2 antibody (19).Details included below.
Mouse species and adenovirus
By tail vein injections by adenovirus (1X108A plaque formation unit (pfu) GFP, calcium tune nerve phosphoric acid Enzyme, InsP3R1, InsP3R1DM (S1589A/S1756A), non-specificity (US) RNAi, calcineurin RNAi, Insp3r1RNAi、Insp3r2RNAi、Insp3r3RNAi、Crtc2RNAi、1X109Pfu CRE-luc reports body, 5X107pfu RSV β-gal) it is delivered to 8-10 week old males C57BL/6J, B6.V-lep<ob>/J,B6.Cg-m+/+Lepr<db>In/J (17).It has been previously described Insp3r2 knock-out mices (10).Make before research all mouse all adapt to their environment 1 week and In temperature controlled environment when 12 is small under illumination/dark cycle drylot feeding in group's cage.It is tested for in-vivo imaging, in gland Is imaged mouse in 3-5 days after Viral delivery.Wild type CRTC2, CRTC2 (S171A), GFP, non-specificity previously had been described RNAi, Crtc2RNAi, CRE-luc and RSV β-gal adenovirus (17,20).Contain rat from the generation of InsP3R1 plasmids The adenovirus of InsP3R1, InsP3R1DM and InsP3R1 (S2682A).Use mouse calcineurin plasmid (Addgene) calcineurin adenovirus is built.CRTC2 Δs CBD (51-692aa), S275A and S171A/S275A adenopathies Poison is made by mouse CRTC2.With the mouse for being blended in the N-terminal myristoylation label (MGSSKSKPKDPSQR) from Src CRTC2 generates myristoylation CRTC2 (Myr-CRTC2) adenovirus.Respectively using sequence 5 '- GGGTACCGCATGTACAGGAAAA-3’、5’-GGGTACTGGAATAGCCTCTTCC-3’、5’- GGGTAACAAGCACCACCATCCC-3 ' and 5 '-GGGCAAGCTGCAGGTGTTCCTG-3 ' structure calcineurins RNAi, Insp3r1 RNAi, Insp3r2 RNAi, Insp3r3 RNAi adenovirus.All expression used in this research Construct is all confirmed by sequencing.
Analysis in vivo
For in-vivo imaging, such as described (17,20) after under the conditions of arbitrary feeding or when fasting 6 is small by mouse into Picture.For pyruvic acid excitation experiment, by mouse overnight fasting and with pyruvic acid (2g kg-1) the interior injection of peritonaeum.Use LifeScan Automatic saccharometer measures blood glucose value.For Western blotting, mouse tissue, centrifugation and reservation supernatant are ultrasonically treated to carry out albumen Matter measures and SDS-PAGE analyses.
Cell culture, cell grade separation, luciferase assay, Calcineurin Activities and cAMP measurements
Containing 10% FBS (HyClone), 100 mg ml–1HEK293T is cultivated in the DMEM of Pen .- Strep (ATCC) cell.Separation and culture mouse primary hepatocytes (18) as discussed previously.Cell grade point is carried out as reported previously From research (18).For report body research, the liver cell (each 1 pfu of cell) that Ad-CRE-luc infects is made to be exposed to the high blood of pancreas When sugar element (Gcg, 100 nM) 2 to 4 are small.It can for cyclosporin A (CsA, 10 μM) or okadaic acid (OA, 100 nM) or cell Permeate the self inhibitory peptide of calcineurin (10 μM) or CN585 (100 μM) or Calyculin A (Calyculin A) (10 nM) or smooth Spongistatin C (Xc, 2 μM) or H89 (30 μM) or (50 μM) inhibition of BAPTA, are used When inhibitor pretreatment liver cell 1 is small.Betagalactosidase activity compared with the RSV β-gal of adenovirus coding makes fluorescein Enzymatic activity standardizes.According to manufacturer specification, (test kit comes from Enzo Life to measurement Calcineurin Activities ) and cell cAMP levels (test kit come from Cayman Chemical Company) Sciences.
Calcium is imaged
Mouse primary hepatocytes are coated on glass cover-slip and containing 0.025% (w/v) Pu Luonike (pluronic) with 5 μM of Fura-2 in the presence of the culture medium 199 (Mediatech) of F127 (Sigma-Aldrich) Acetoxymethyl (Molecular Probes) loads 30 minutes.Coverslip is placed in laminar flow perfusion compartment (Warner Instruments Corp.) on and the infusion with 199 or 100 nM glucagons of culture medium in culture medium 199. Excitation wavelength collects the image for the cell that Fura-2 is loaded with cooling CCD camera under replacing between 340 nm and 380 nm. The fluorescence intensity ratio under two kinds of excitation wavelengths is calculated after background fluorescence is subtracted.Calibrating reagent box is imaged using Fura-2 calcium (Invitrogen) [Ca2+] I (cytosol free calcium concentration) is calculated.Use MetaFluor software packages (Universal Imaging Corp.) collect and analyze image.Figure represents to be obtained by representative single test indivedual from each group 30-40 The average response of cell.Bar chart represents that average response of each condition from 150-200 cell (is more than times of average baselining Number).All experiments all repeat at least 3 times, obtain similar results.
Western blotting, immune precipitation, immunostaining
As the progress Western blotting, immune precipitation and immunostaining measure (18).It is previously described CRTC2, pCREB (Ser133), CREB, pAKT (Thr308), AKT, tubulin, HA and FLAG antibody (18).Anti- InsP3R1 (the A302- of antibody It 158A) is bought with InsP3R3 (A302-160A) from Bethyl laboratories, anti-InsP3R2 (ab77838) comes from Abcam, anticalcium Adjusting neural phosphatase (610260), anti-GRP78 (ADI-SPA-826-F) comes from Enzo Life from BD Biosciences Sciences, anti-phosphoric acid PKA substrates (RRXS/T, 9624), anti-phosphoric acid AKT substrates (RXXS/T, 9614) and CRTC2 (pS171, 2892) Cell Signaling are come from.CRTC2 (pS275) antibody (19) is used as described.
Quantitative PCR
The total cell RNA and use from entire liver or from primary hepatocyte are extracted using RNeasy kits (Qiagen) In with SuperScript II enzymes (Invitrogen) generation cDNA.As described, pass through quantitative PCR analysis cDNA (18).
Mass spectroscopy
Prepare the endogenous CRTC2 from HEK293T cells for mass spectroscopy research (21) as reported previously Immune precipitation and from glucagon stimulate liver cell the sero-fast immune precipitation of phosphoric acid PKA substrates, and by Electrospray ionization tandem mass spectroscopy is carried out on Thermo LTQ Orbitrap instruments to be analyzed.
Statistical analysis
All researchs are all carried out at least three independence occasion.As a result it is reported as average value ± s.e.m.It is unworthy of using double tails The comparison of different groups of progress is examined to student t.In P<Under 0.05, difference is considered as with statistical significance.
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Embodiment 10：For treating and preventing p38 the and MK2/3 inhibitor of metabolic disease
The detrimental effect that the downstream of CaMKII can mediate CaMKII there are two kinds kinases-p38 and MK2 (referring to figure 51).More multipath can be covered although blocking the drug of the more upstream target such as IP3 receptors and CaMKII, they can also have more Toxicity.On the contrary, targeting further downstream mediators undershooting-effect can be caused smaller.MK2 is extremely similar with MK3, and blocks the reagent of MK2 (DN-MK2 and MK2 inhibitor) blocks both MK2 and MK3.If only block one of they, then another one will play compensation Effect.
Therefore, p38 inhibitor and MK2/3 inhibitor can also be used for the metabolic disease that treatment is induced by obesity, such as 1 type sugar Urinate disease, diabetes B, insulin resistance and metabolic syndrome.
Research described in Figure 52-56 uses primary hepatocytes (HC).For p38 test, labeled as " Cre " or " gland- P38 in the sample of Cre " or " ad-Cre " lacked and this control be gland-LacZ (sometimes labeled as ad-LacZ or LacZ).(HC has a form of p38 genes, is lacked when exposed to the Cre recombinases delivered using adenovirus vector It loses.Control is the adenovirus vector for the irrelevant protein that coding is known as LacZ.)
Figure 52 directly makes the crucial distal end consequence of p38 and CaMKII, i.e. FoxO1 nuclear locations are related：Normal existing for p38 In HC (LacZ), constitutive activity (CA) CaMKII promotes FoxO1 to enter in core, but in the HC (Cre) for lacking p38, it is not It can so do.
Figure 53 shows MK2 in core FoxO1 with inducing both genes involved in hepatic glucose generation using MK2 inhibitor In importance.MK-2 inhibitor comes from Calbiochem (catalog number (Cat.No.) 475864)：2- (2- quinoline -3- yl pyridines -4- bases) -1, 5,6,7- tetrahydrochysene -4H- pyrrolo-es-[3,2-c] pyridine -4- ketone.
Figure 54 using simulation wherein liver cell be subjected to endoplasmic reticulum (ER) stress obesity and diabetes B in process Experimental model (the protein Misfolding derivant for being known as tunicamycin (tunicamycin)).ER stress be by ' PERK/PKR' and ' ATF4/CHOP'(lower parts right-hand branch) it represents (referring to Figure 51).P38 is shown in Figure 54 for tunicamycin (ER stress) induction P-PERK (its activity form) and CHOP increase needed for.Also it will be observed that in the HC (Cre) for lacking p38, p58IPK is omited Micro- but consistent rise-this also complies with the lower part right-hand branch of Figure 51.
Figure 55 using simulation obesity and diabetes in liver ER stress another experimental model (be known as palmitate fat Fat acid).In Figure 54, p38 is needed for CHOP and p-PERK increase.
If Figure 56 watches the bottom of the right-hand branch of Figure 51 attentively, then it will be observed that reading is sound of the p-Akt to insulin It should.All HC in Figure 56 receive the insulin of acute dose to test the ability of Insulin activation (phosphorylation) Akt.Herein Model for simulating obesity/diabetes is the palmitate model used in Figure 55.It can be observed in the normal HC of p38 (Ad-LacZ) in, insulin is given strong p-Akt signals (preceding 2 swimming lanes) but this and is reduced by palmitate (" palm ").It faces Situation about occurring in this " insulin resistance " simulation obesity and diabetes B of palmitate.If then watch no p38 attentively HC (Ad-Cre), then the ability of Insulin activation Akt (p-Akt) is improved.This and the p38-p-Akt shown in Figure 51 Association is consistent.
Figure 86,87 and 88, which are related to, inhibits how CaMKII, p38 or MK2/3 cause insulin signal transduction to improve, i.e. this How a little inhibitor prevent insulin resistance.Wherein data support following mechanism：The inhibitor of CaMKII, p38 or MK2/3 reduce Tribble3 (Trb3), this then improve insulin make AKT phosphorylations ability (Trb3 be AKT phosphorylations endogenous inhibit Agent).
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Claims (7)

1. reduce the examination of the activity of the protein kinase 3 (MK2/3) of the protein kinase 2/MAP kinase activations of map kinase activation Agent is preparing to treat or prevent the purposes in the drug of the diabetes B of subject, insulin resistance or metabolic syndrome.
2. purposes as described in claim 1, wherein described treat or prevent the decomposition of glycogen or sugared different for influencing the subject It is raw.
3. purposes as described in claim 1, wherein described treat or prevent the hepatic glucose generation for reducing the subject, height The relevant inflammation of blood glucose, hyperinsulinemia, fatty liver, insulin resistance, insulin resistance, the relevant blood fat of insulin resistance Abnormal or any combination thereof.
4. purposes as described in claim 1, wherein described treat or prevent includes reducing coding institute to subject application The step of stating the antisense RNA of the expression of the gene of MK2/3 protein.
5. purposes as described in claim 1, wherein described treat or prevent includes specifically binding to subject application The step of peptide or polypeptide of the MK2/3 protein.
6. purposes as described in claim 1, wherein described treat or prevent the step for including that small molecule is applied to the subject Suddenly.
7. purposes as claimed in claim 6, wherein the small molecule is MK2/3 inhibitor.
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